Chemico-Biological Interactions 184 (2010) 484491

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Chemico-Biological Interactions
j our nal homepage: www. el sevi er . com/ l ocat e/ chembi oi nt
Protective effects of pre-germinated brown rice diet on low levels of Pb-induced
learning and memory decits in developing rat
Rong Zhang
a
, Hongzhi Lu
a
, Su Tian
a
, Jie Yin
b
, Qing Chen
a
, Li Ma
a
, Shijie Cui
a
, Yujie Niu
a,
a
Department of Occupational Health and Environmental Health, School of Public Health, Hebei Medical University, Zhongshan East Road 361,
Shijiazhuang 050017, Hebei, Peoples Republic of China
b
Department of Gynaecology and Obstetrics, The Fourth Hospital of Hebei Medical University, Healthy Road 12, Shijiazhuang 050011, Hebei, Peoples Republic of China
a r t i c l e i n f o
Article history:
Received 4 December 2009
Received in revised form 28 January 2010
Accepted 28 January 2010
Available online 6 February 2010
Keywords:
Lead (Pb)
Pre-germinated brown rice
Developing nervous system
GABA
Oxidative 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 low
levels of Pb show learning and memory impairments. Unfortunately, Pb-poisoning cases continue to
occur 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 were
randomly divided into ve groups: the control group (AIN-93G diet, de-ionized water), the lead acetate
(PbAC) group (AIN-93G diet, 2g/L PbAC in de-ionized water), the lead acetate +WR group (white rice
diet, 2g/L PbAC in de-ionized water; PbAC+WR), the lead acetate +BR group (brown rice diet, 2g/L PbAC
in de-ionized water; PbAC+BR) and the lead acetate +PR group (pre-germinated brown rice diet, 2g/L
PbAC in de-ionized water; PbAC+PR). The animals received the different diets until PND60, and then the
experiments were terminated. The protective effects of pre-germinated brown rice (PR) on Pb-induced
learning and memory impairment inweaning 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 were
evaluated. The levels of reactive oxygen species (ROS) were determined by ow cytometry. The levels of
8-hydroxy-2-deoxyguanosine (8-OHdG), -aminobutyric acid (GABA) and glutamate were determined
by HPLC. Our data showed that feeding a PR diet decreased the accumulation of lead and decreased
Pb-induced learning and memory decits in developing rats. The mechanisms might be related to the
anti-oxidative effects andlarge amount of GABAinPR. Our studyprovides a regimentoreduce Pb-induced
toxicity, especially future learning and memory decits in the developing brain.
2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Pb is an environmental neurotoxicant known to produce detri-
mental effects in the nervous system. Numerous studies have
provided evidence that animals chronically intoxicated with low
levels of Pb show cognitive decits [13]. Epidemiological analysis
indicatedthat childrenexposedtolowlevels of Pbare at highrisk of
learning and memory impairments [4]. Morphologically, Pb affects
axonal and synaptic elaboration; neurochemically, it replaces cal-
cium resulting in altered calcium channel and NMDA receptor
function; and metabolically, it causes oxidative stress and damages
Abbreviations: AP, alkaline phosphatase; BR, brown rice; 2

-dG, 2

-
deoxyguanosine; EDTA, ethylenediamine tetraacetic acid; GABA, -aminobutyric
acid; Glu, glutamate; HPLC, high-performance liquid chromatography; NP1, nucle-
ase P1; PR, pre-germinated brown rice; ROS, reactive oxygen species; WR, white
rice; 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).
the developing brain [5,6]. In many countries Pb-poisoning cases
continue 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 been
explored. However, there are no randomized, clinical trials of
Pb-poisoned children providing evidence that treatment with
chelators improves clinical outcomes, particularly future cogni-
tive development. In large population studies, children who have
dietary deciencies in iron, calcium, vitamin C, or zinc are more
susceptible to injury from environmental sources of lead, whereas
preschool urban children who have higher dietary iron intake have
lower blood lead levels [9]. Therefore, treatment regimens have
focused on reducing Pb-induced toxicity especially future learning
and memory [10].
The Pb-induced impairment of synaptic plasticity and its
effects on learning and memory decits have been adequately
studied. Previous studies suggested that oxidative stress, intracel-
0009-2797/$ see front matter 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2010.01.043
R. Zhang et al. / Chemico-Biological Interactions 184 (2010) 484491 485
lular calcium, long-term potentiation, the specic roles of GABA,
the metabotropic glutamate receptor, the cholinergic nicotinic
receptor and nitric oxide are related to Pb exposure-induced devel-
opmental neurotoxicity including learning and memory defects
[11]. These toxic mechanisms have led scientists to study the
protective qualities of regimens with anti-oxidation and other pro-
tective mechanisms on lead toxicity. White rice (WR), the staple
food of Asians, is manufactured by eliminating the ber-rich bran
layer fromunpolished rice, that is, brown rice (BR). Pre-germinated
brown rice (PR) has been developed by soaking BR in water to
induce slight germination. After germination, there is the advan-
tage that PR does not have the hardness of brown rice. More
importantly, following germination, the levels of several valuable
nutrients within the grain increase, especially oryzanol and -
aminobutyric acid (GABA). The quantity of nutrients contained in
PR compared to milled rice are 13 times for oryzanol, 10 times for
GABA, nearly 4 times for dietary ber, vitamin E, niacin and lysine,
and about 3 times for vitamin B1, B6 and magnesium. Previous
research has demonstrated that the benecial effects of abundant
phenolic compounds in PR have been attributed mainly to their
anti-oxidant activities [12]. It has recently been reported that PR
facilitates spatial learning, and therapeutically, PR may prevent
Alzheimers disease (AD) [13]. However, it remains to be claried
if these nutrients improve brain function, particularly learning and
memory abilities in the developing brain after Pb exposure.
Therefore, the effects of feeding three rice diets (WR, BR and
PR) on learning and memory impairment induced by Pb in wean-
ing rats were evaluated in this study. The phenolic compounds in
PR are known to have anti-oxidant properties, while developmen-
tal exposure to Pb has been shown to be elevated in AD and is
known to generate reactive oxygen species (ROS) in the aging brain
[14]. Therefore, we examinedwhether feedingdevelopingrats with
different rice diets can counter the oxidative damage induced by
Pb. Our results showed that feeding a PR diet could decrease the
accumulation of lead and improve learning and memory decits
in developing rats after Pb exposure. These mechanisms might be
related to the anti-oxidative effects of phenolic compounds and
the large amount of GABA in PR. This study provides a regimen to
reduce Pb-induced toxicity, especially future learning and memory
in developing brain.
2. Materials and methods
2.1. Animals and foods
SpragueDawley rats (75 males and 75 females; 486g) at the
weaning age of 21 days (PND21) were procured from the Experi-
mental Animal Center of Hebei Medical University (Shijiazhuang,
China) and housed in polycarbonate cages with lter tops. Ani-
mals were maintained under standard conditions of temperature
(252

C), relativehumidity(502%), andnatural lightdarkcycle

for 1 week to acclimate. All animals were fed a diet and water
ad libitum in stainless cages, and they received humane treatment
in compliance with the Principles of Laboratory Animal Care for-
mulated by the National Society for Medical Research and the
Guide for the Care and Use of Laboratory Animals prepared by
the National Academy of Sciences and published by the National
Institutes of Health (NIH Publication No. 80-23, revised 1978). The
ethical regulations have been followed in accordance with national
and institutional guidelines for the protection of animal welfare
during experiments.
All rice and control diets were manufactured as pellets by the
Experimental Animal Center of Chinese Academy of Medical Sci-
ences (Beijing, China). The control diet (AIN-93G) was composed
of cornstarch [39.7% (w/w)], -cornstarch [13.2% (w/w)], casein
[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, or
WR diets were produced by replacing cornstarch and -cornstarch
withpre-germinatedbrown, brown, or whiterice, respectively[15].
The weaning rats were randomly divided into 5 groups (30 rats
per treatment group): the control group (AIN-93G diet, de-ionized
water), the lead acetate (PbAC) group (AIN-93G diet, 2g/L lead
acetate in de-ionized water), the lead acetate +WR group (white
rice diet, 2g/L PbAC in de-ionized water; PbAC+WR), the lead
acetate +BR group (brown rice diet, 2g/L PbAC in de-ionized water;
PbAC+BR) and the lead acetate +PR group (pre-germinated brown
rice diet, 2g/L PbAC in de-ionized water; PbAC+PR).
On PND60, six animals per group were used for Morris water
maze test, and other six animals per group were used for passive
avoidance test. The remaininganimals were decapitatedafter being
anesthetized by ether for further assessing the biochemical param-
eters. Sixanimals per groups were usedfor oxidative stress markers
(GSH-Px, SOD, MDA and 8-OHdG levels), while other six animals
per group were used to determine the levels of lead, ALA, GABA
and glutamate. The remaining six animals per group were used to
determine the ROS levels.
After the animals were anesthetized, blood samples were col-
lected fromfemoral artery. Serumwas harvested by centrifugation
at 700g for 10min. Brains were removed and placed on an ice-
cold plate. The hippocampus was excised from the undersurface
of the corpus callosum for ROS measurement or stored in liquid
nitrogen for further analysis.
2.2. Determination of lead levels
To identify the levels of lead exposure of each litter, the lead
concentrations in the sera and hippocampi were measured by
atomic absorption spectrophotometry (AAS, TAS-990AFG, Beijing
Purkinje General Instrument Co., Ltd., Beijing, China). Hippocampi
were taken out from liquid nitrogen and thawed. About 0.10.3g
of each tissue were weighed, digested and analyzed for lead con-
tent. Briey, prior to elemental analysis, the tissues were digested
with nitric acid (ultrapure grade, Beijing Fine Chemical Ltd., Bei-
jing, China) in a polytetrauoroethylene (PTFE) vessel sealed in a
steel vessel overnight. After adding 0.5mL of H
2
O
2
, the mixed solu-
tions were completely digestedby the microwave digestionsystem
(MDS, Sineo Microwave Chemistry Technology Co., LTD., China).
Then, the solutions were heated at 120

C to remove the remaining

nitric acid until the solutions were colorless and clear. The remain-
ing solutions were diluted to 3mL with 2% nitric acid. Then, the
lead concentration in serum and digested hippocampal samples
was detected at 283.3nm.
2.3. Determination of learning and memory
2.3.1. Morris water maze test
On PND60, animals were tested for their spatial memory by the
Morris water maze. The task was adapted fromthe paradigmorigi-
nally describedinthe literature [16]. Briey, a circular tank (180cm
in diameter 60cmin height) was lled with water (261

C) to a
depth of 40cm, and the water was made opaque with milk powder
to prevent visualizationof the platform(10cm10cm), whichwas
submerged1.5cmunder thewater surface. Roomlights illuminated
the pool, and multiple distant cues around the room(window, cab-
inets, and furniture) were kept in the same location throughout
the 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
486 R. Zhang et al. / Chemico-Biological Interactions 184 (2010) 484491
the pool was xed in the middle of the perimeter of the pool (N, E, S
and W). Rats were trained from PND61 to PND65 during ve daily
trials. At the beginning, animals were individually placed into the
pool facing the wall. If the rat failed to reach the platform in 60s,
it was gently guided to the platform. Once on the platform, the
rat was left there for 30s. Between trials, the rats were toweled,
fan dried and kept in holding cages for at least 5min. On PND66,
escape latencies from the three quadrants were recorded as their
nal performances.
2.3.2. Passive avoidance test
Animals from each group were raised on PND60 to detect
their learning and memory abilities by one-trial-learning passive
avoidance test. A step through the type one-trial-learning passive
avoidance test was used as described earlier [17]. A rectangular
box (50cm20cm30cm) consisted of a dark and an illuminated
compartment equipped with a grid oor. The two compartments
were connected by a little door that allowed the rats pass through.
During passive avoidance conditioning, the illuminated compart-
ment was the only light source in the room. On day 1, after
habituation to the dark compartment for 2min, the rat was placed
in the illuminated compartment and allowed to enter the dark
compartment. Upon entry, the door was closed, and the animal
remained in the dark compartment for 10s. Thereafter, the rat
was placed back in its home cage. Three such training trails were
also run on day 2, and the latency to enter the dark compartment
was scored. During the third learning trial the rat received a single
scrambled electric foot shock of 0.5mA for 2s when the rat entered
the darkcompartment. The rat was removedafter receivingthe foot
shock and returned to the light compartment by the experimenter.
The door was re-opened 10s later to start the next trial. Training
continued in this manner until the rats stayed in the light compart-
ment for more than 120s in a single trial. Twenty-four hours later,
each animal was placed in the light compartment, and the step-
through latency was recorded until 300s had elapsed (retention
trial).
2.4. Determination of GABA and glutamate levels
GABA and glutamate levels were quantied by high-
performance liquid chromatography (LC-20AT, Shimadzu
Corporation, Japan) with a uorescence detector (RF-10AXL).
Samples were separated on a Hypersil C18-BDS HPLC column
(4.6mm250mm in length and 10m beads).
Briey, the hippocampal samples were taken out from liquid
nitrogen and homogenized in 2.4mL 0.04mol/L of perchloric acid,
andthencentrifugedat 10,000g for 20min. The 1mL supernatant
was neutralized with 1mL of 1.5mol/L of KHCO
3
and then ltered
through a 0.22m millipore lter. Twenty microliters of the hip-
pocampal samples were mixedwith80L of methanol. Then, 20L
of the mixture was added to 20L of OPA-MCE reagent, which
was obtained by mixing 25mg of OPA (o-phthaldialdehyde, Sigma)
with 0.5mL of methanol and 50L of -mercaptoethanol (-MCE,
Sigma) in 5.0mL of boric acid buffer (pH 9.6). After 2min, 20L of
this mixture was injected into an HPLC column. The mobile phase
consisted of 50mmol/L of sodium acetate and methanol (55:45,
v/v) adjusted to pH 6.0. The mobile phase was ltered through
a 0.22m millipore lter and ultrasound degassed prior to use.
Chromatographic analyses were performed at 252

C. The ow
rate was 0.8mL/min. Amino acids were detected uorometrically
at an excitation wavelength of 340nmand an emission wavelength
of 450nm. Glutamate was eluted with a peak at 2.8min, followed
by GABA at 9.5min. Standard formulations of GABA and glutamate
were purchased from Sigma. All drugs were made up immediately
prior to the experiment and were applied to the bath.
2.5. Measurement of oxidative stress state
2.5.1. ROS evaluations in hippocampus cells
The intracellular ROS was detected using 2

,7

-
dichlorouorescein diacetate (DCFH-DA) as a probe [18]. DCFH-DA
is hydrolyzed by esterase to dichlorouorescein (DCFH), which
is trapped within the cell. This nonuorescent molecule is then
oxidized to uorescent dichlorouorescein (DCF) by the action
of cellular oxidants. DCFH-DA cannot be appreciably oxidized to
a uorescent state without prior hydrolysis. In order to retain
the activities of esterase and the oxidized action of DCFH by the
action of cellular oxidants within the cell, the acutely isolated
hippocampal cells were used for ROS measurement. Rats were
sacriced under sterile conditions, and the hippocampi were
excised. The samples were rapidly dissected into small pieces and
incubated for 20min at 37

C in a solution of 2.5mg/mL trypsin in

Ca
2+
- and Mg
2+
-free Hanks balanced salt solution (HBSS) buffered
with 10mM HEPES. After letting the tissue fragments settle, the
supernatant was collected and one-tenth volume of fetal bovine
serum (FBS) was added to stop the trypsin action. The supernatant
was stored in ice. The remaining tissue fragments were redigested
for an additional 20min at 37

C. Then the resulting cell suspen-

sion was combined and centrifuged for 5min at 800g after
ltering with 200 mesh copper grids. The cells were resuspended
in DMEM medium with 10% FBS, 0.5mM l-glutamine, 100U/mL
penicillin and 100U/mL streptomycin. The cells were then dis-
tributed to six-well plastic culture plates (Costar, Cambridge,
MA) at approximately 1.010
6
cells per well in 2mL of culture
medium. Then, the cells were incubated at a nal concentration
of 10mM DCFH-DA (Bryotime Institute of Biotechnology, China)
for 20min at 37

C in 95% air and 5% CO

2
. Cells were maintained
in the dark prior to and during the analysis. As soon as the incu-
bation was completed, the samples were placed on ice for 5min
to stop the reaction. The formation of the uorescent-oxidized
derivative of DCF was monitored using a FACS420 ow cytome-
ter (Becton Dickinson, USA) at emission wavelength of 525nm
and excitation wavelength of 488nm. Finally, ROS generation
was quantied by the median uorescence intensity of 10,000
cells.
2.5.2. Quantication of GSH-Px and SOD activities as well as MDA
levels
The activities of anti-oxidase glutathione peroxidase (GSH-
Px) and superoxide dismutase (SOD), and the levels of the lipid
peroxidation (LPO) product malonaldehyde (MDA) in sera and hip-
pocampi were determined according to the methods described
in the references using commercial kits (Nanjing Jiancheng Bio-
eng Inst., China) [19]. To prepare tissue extracts, the hippocampi
were ground with liquid nitrogen in a mortar, and the ground
tissues (0.5g each) were then treated with 4.5mL of PBS buffer.
The mixtures were homogenized using a glass homogenizer for
1min on ice. The homogenates were then ltered and centrifuged
using a refrigerated centrifuge at 4

C. Then, these supernatants

were used to determine the enzymes activities using a microplate
reader (Synergy
TM
HT, BioTek Instruments, Inc., USA). The pro-
tein levels of samples were measured by the Coomassie Brilliant
Blue G-250 method with bovine serum albumin as standard. The
protein contents were determined according to the manufac-
turers instructions (Nanjing Jiancheng Bioeng Inst., China). The
data expressed as units of activity or nanomoles per milligram of
protein.
2.5.3. Measurement of 8-OHdG and 2

-dG
Levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) were quan-
tied according to the previous study described with a minor
modication [20]. Briey, DNA was extracted from hippocampi by
R. Zhang et al. / Chemico-Biological Interactions 184 (2010) 484491 487
homogenization in buffer containing 1% sodium dodecyl sulfate,
10mMNaCl, 10mMEDTA, and a 3-h incubation in 100g/mL pro-
teinase K at 50

C. Homogenates were extracted with Trisphenol

and trichloromethane. The extracts were mixed with 3M sodium
acetate and 2vols of 100%ethanol to precipitate DNAat 20

C. The
samples were washed twice with 70% ethanol, air-dried for 15min
and dissolved in 100L of 10mM Tris/1mM EDTA (pH 8.0). Then,
DNAwas digestedwith10unit/L of nuclease P1(NP1, Biolong Bio-
logical and Technology Co., Ltd., Shanghai, China) and 1 unit/L of
alkaline phosphatase (AP1, Takara Biotechnology CO., LTD., Dalian,
China). The digest was ltered through a 0.22m millipore lter,
and then 8-OHdG and 2-deoxyguanosine (2

-dG) levels were mea-

sured using high-performance liquid chromatography (LC-20AT,
Shimadzu Corporation, Japan) with a UV-detector (SPD-20A). Stan-
dard 8-OHdGand 2

-dGwere purchased fromSigma. Samples were

separated on a Hypersil C18-BDS HPLC column (4.6mm250mm
in length and 10m beads). Twenty microliters of DNA digest
was injected into the HPLC column. The mobile phase consisted of
50mmol/L of KH
2
PO
4
and methanol (90:10, v/v) adjusted to pH5.5.
The mobile phase was ltered through 0.22mmillipore lter and
ultrasound degassed prior to use. Chromatographic analyses were
performed at 252

C. The ow rate was 1mL/min. 8-OHdG and

2

-dG were detected at a wavelength of 260nm. 2

-dG was eluted

with a peak at 8.4min, followed by 8-OHdG at 12.2min. Levels of
8-OHdG were expressed as 8-oxo-dG/10
5
dG.
2.6. Determination of -aminolevulinic acid (ALA) levels in sera
The -aminolevulinic acidconcentrationwas measuredbya col-
orimetric method according to the report of Tomokuni and Ogata
with a small modication [21]. Briey, 200L of serum was added
to 800L of PBS. Then, 1.0mL of the mixture was added into each
of two 10-mL glass-stoppered tubes, and 1.0mL of acetate buffer
(pH 4.6) was added. To one of the tubes, 200L of ethyl acetoac-
etate was added and mixed with a vibration mixer for about 5s.
Ethyl acetoacetate was omitted from the other tube, which was
run as a blank. The tubes were placed in a boiling water bath for
10min. After cooling, we added 3.0mL of ethyl acetate and shook
the tubes 50 times by hand to extract the ALA-pyrrole. After cen-
trifuging for 3min at 15002000g, we pipetted 2.0mL of the
ethyl acetate (upper) layer into another glass tube. Then, we added
2.0mL of modied Ehrlichs reagent and mixed the solution. After
10min, we determined the absorbance of the color solution at
553nm. To calculate the concentration of ALA, we measured the
absorbance of serum with (A) and without ethyl acetoacetate (B),
subtracted the absorbance of B from the absorbance of A, and read
the concentration of ALA from a standard curve prepared from
an aqueous solution of ALA. Standard ALA was purchased from
Sigma.
2.7. Statistical analysis
The gure legends show the means SDs. Data were analyzed
using one-way analysis of variance (ANOVA) followed by the post
hoc Dunnetts test when F was signicant. Differences were con-
sidered signicant when p<0.05.
3. Results
3.1. Pb levels after feeding different diets to rats
The Pb levels in sera and hippocampi were elevated after Pb
exposure (p<0.05, Fig. 1). The lead levels in sera and hippocampi
of rats both in the PbAC+BR and PbAC+PR groups were decreased
signicantly 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 were
expressed as meanSD (n=6). The Pb levels in sera and hippocampi of rats after
PbAC treatment were signicantly increased compared with the control (*p<0.05),
respectively. The Pb levels in sera and hippocampi of rats on BR and PR diet were
decreased signicantly 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 memory
abilities
Learning and memory abilities were evaluated by the Morris
water maze and positive avoidance test. Fig. 2 shows the perfor-
mance in the Morris water maze test. The escape latencies of rats in
the PbACandPbAC+WRgroups were slowlyshortenedbyrepeated
training, whereas those of the control, PbAC+BR and PbAC+PR
groups were rapidly shortened (Fig. 2A). The nal 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 nal
test. Data were expressed as meanSD escape latency (s) onto a submerged plat-
form (n=6). (A) Latency to nd the platform during training sessions. (B) Latency
examined 24h after the last training session. The escape latencies of rats in both
PbAC and PbAC+WR groups were increased signicantly compared with the con-
trol (*p<0.05). The escape latencies of rat in both PbAC+BR and PbAC+PR groups
were reduced signicantly compared with that of the PbAC group (

p<0.05).
488 R. Zhang et al. / Chemico-Biological Interactions 184 (2010) 484491
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 meanSD
step-through latency (s) onto a light box (n=6). The step-through latencies of rats
in PbAC, PbAC+WR and PbAC+BR groups were increased signicantly compared
with the control (*p<0.05), respectively. The step-through latencies of rats in both
PbAC+BR and PbAC+PR groups were reduced signicantly compared with that of
the PbAC group (

p<0.05), respectively.
formed24hafter the last training session. The nal escape latencies
of rats both in the PbAC and PbAC+WR groups were increased sig-
nicantly compared with that of the control group (p<0.05). In
addition, the nal escape latencies of rats both in the PbAC+BR
and PbAC+PR groups were reduced signicantly compared with
that of the PbAC group (p<0.05; Fig. 2B).
Fig. 3 shows the results of performance in the step-through
type passive avoidance test. There were signicant increases in the
step-through latencies of rats exposed to lead acetate (in the PbAC,
PbAC+WR and PbAC+BR groups) in the retention trial compared
with that of the control group (p<0.05). The step-through latencies
of rats in both the PbAC+BR and PbAC+PR groups were reduced
signicantly compared with that of the PbAC group (p<0.05).
3.3. Effects of feeding different diets on GABA and glutamate
content in rat hippocampi
The levels of GABA and glutamate in hippocampi were mea-
sured with HPLC (Fig. 4). After Pb treatment, the GABA content was
signicantly decreased compared with the control (
*
p<0.05). The
GABAlevels inhippocampi fromrats inthe PbAC+BRandPbAC+PR
groups weresignicantlyincreasedcomparedwiththat of thePbAC
group (p<0.05). The glutamate levels in rat hippocampi from the
PbACgroupwere signicantlyincreasedcomparedwiththe control
(p<0.05).
Fig. 4. The levels of GABA and glutamate in hippocampi. Data were expressed as
meanSD (n=6). After Pb treatment, the GABA levels were signicantly decreased
compared with the control (
*
p<0.05). The GABA levels in hippocampi of rats in
PbAC+BR and PbAC+PR groups were signicantly increased compared with PbAC
group (

p<0.05), respectively. The glutamate levels in hippocampi of rats in PbAC

group were signicantly increased compared with the control (
*
p<0.05).
Fig. 5. Effects of feeding different diets on Pb-induced ROS levels by owcytometry
using the DCFH-DA probe. Data were expressed as meanSD(n=6). The ROS levels
of rats in PbAC group were signicantly different compared with that of the con-
trol (*p<0.05). The ROS levels of rats with different rice diet (PbAC+WR, PbAC+BR
and PbAC+PR) were decreased signicantly 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 of
anti-oxidants results in oxidative stress in biological systems. Fig. 5
shows that the ROS levels in hippocampal cells of PbAC-treated
rats on the AIN-93G diet were signicantly higher compared with
the control, whereas the ROS levels were signicantly decreased
in hippocampal cells of rats on the rice diets (PbAC+WR, PbAC+BR
and PbAC+PR groups) compared with that of the PbAC-treated rats
on the AIN-93G diet (p<0.05). These results indicated that feeding
the rice diets might prevent ROS generation in the hippocampi of
Pb-exposed rats.
ROS could react with lipids to generate MDA, which could
form DNA adducts and cause oxidative cell damage [22]. In this
study, we observed signicantly increased MDA levels in sera
from 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 signicantly in
the PbAC+PR group compared with the control. The MDA levels in
sera from PbAC+WR-, PbAC+BR- and PbAC+PR-treated animals
were reduced signicantly compared with that of the PbAC group
(p<0.05), but no signicant changes were seen in the hippocampi
from rats on the different diet groups. These results indicated that
the rice diet played animportant anti-oxidative role onPb-exposed
rats, especially the PR diet.
Studies suggested that one of the important mechanisms asso-
ciated with toxic effects of lead is oxidative stress caused by
disruption of the oxidant/anti-oxidant balance in animals includ-
ing humans. Reduced levels of glutathione (GSH) and activities of
GSH-Px and SOD in tissues or blood are most commonly used to
evaluate lead-induced oxidative damage [22]. In this study, the
GSH-Px and SOD activities in sera and hippocampi from PbAC-
treated animals fed with different diets were decreased compared
with those of the control (Table 1). The GSH-Px and SOD activities
in sera of rats in PbAC treatment groups feeding different rice diets
(PbAC+WR, PbAC+BR and PbAC+PR) were signicantly increased
(p<0.05) compared with those of PbAC-fed animals. The SODactiv-
ities in hippocampi of rats in the PbAC+BR and PbAC+PR groups
were signicantly 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 the
enzyme ALA dehydratase [23]. In this study, we found that the ALA
levels in sera were signicantly 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) 484491 489
Table 1
Levels 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 0.29
PbAC 230.54 6.82
*
90.28 12.67
*
25.71 1.34
*
1.08 0.18
*
17.36 1.96
*
2.18 0.43
PbAC+WR 253.52 3.38
*
91.74 13.16
*
28.78 0.46
*
1.17 0.26
*
13.93 1.67
*
2.07 0.61
PbAC+BR 269.51 2.82
*
102.41 14.55
*
31.16 0.41
*
6.02 0.89
*
6.06 0.95
*
1.73 0.11
PbAC+PR 277.93 2.03

108.66 17.03
*
29.87 0.76
*
7.95 1.19

3.07 0.29

1.72 0.34
Data represented as means SD (n=6). Values marked with an * are signicantly different from control (p<0.05). Compared with PbAC group,

p<0.05.
Fig. 6. The levels of -aminolevulinic acid levels in sera. Data were expressed as
meanSD (n=6). After Pb treatment, the -aminolevulinic acid levels were signif-
icantly increased compared with the control (
*
p<0.05). The -aminolevulinic acid
levels insera of rats inPbAC+WR, PbAC+BRandPbAC+PRgroups were signicantly
decreased compared with PbAC group (

p<0.05), respectively.
has been shown to cause oxidative damage to DNA in Chinese
hamster ovary cells in vitro through the formation of 8-OHdG
adducts [25,26]. Fig. 7 shows that lead acetate induced a 5.2-fold
increase in the hippocampal 8-OHdG/10
5
2-dG ratio of rats on
the AIN-93G diet (p<0.05), whereas the levels of the hippocam-
pal 8-OHdG/10
5
2-dG ratio were 4.4-, 4.5- and 2.1-fold higher in
rats onthe different rice diets (PbAC+WR, PbAC+BRandPbAC+PR,
respectively) compared with the control. These results suggested
that feeding with the rice diets might resist the formation of 8-
OHdG adducts induced by Pb.
4. Discussion
Behavior, which is the net output of sensory, motor, and cogni-
tive functions in the nervous system, can be a potentially sensitive
endpoint of lead-induced neurotoxicity [27,28]. In previous stud-
ies, lead exposure impaired the ability to complete behavioral tasks
Fig. 7. Comparison of 8-OHdG levels in the hippocampal DNA of rats feeding differ-
ent diets. 8-OHdG relative levels were expressed as the ratio of oxidized DNA base
(8-OHdG) to non-oxidized base (2-dG). Data were expressed as meanSD (n=6).
The 8-OHdG levels in hippocampal DNA of rats in PbAC group were signicantly
increased compared with that of the control (*p<0.05). The 8-OHdG levels in hip-
pocampal DNA of rats with different rice diet (PbAC+WR, PbAC+BR and PbAC+PR)
were decreased signicantly compared with that of PbAC group (

p<0.05), respec-
tively.
in animals [29] and caused a signicant decrease in IQ and other
psychometric scores in children [30]. Our behavioral tests also con-
rmed these results. Compared with the control, the lead-exposed
rats showed a signicant learning decit (Fig. 2). Due to currently
unsuccessful clinical treatments, the regimens of cognitive dys-
function have been a focal point of research efforts. Here, learning
and memory in lead-exposed, weaning rats on different rice diets
were investigated.
We evaluated spatial learning and memory of animals with
the Morris water maze until maturity (PND66). We found the Pb-
exposed rats on different diets took different times to locate the
platform after ve days of acquisition training. The nal escape
latencies of Pb-exposed rats on AIN-93G and WR diets were
increased signicantly. In addition, the nal escape latencies of rats
on BR and PR diets were reduced signicantly compared with that
of the PbAC group (Fig. 1B), whereas there was no statistical differ-
ence in latency of rats on BR and PR diets, compared with that of
the control group. These results suggested that spatial learning and
memory 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 acetate
treatment, we observedsignicantly increasedstep-throughlaten-
cies of Pb-exposed rats on AIN-93G, WR and BR diets in the
retention trial compared with that of the control group. The step-
through latencies of rats on the BR and PR diets were reduced
signicantly compared with that of the PbAC group. Therefore,
feeding BR and PR diets could resist the lead-induced short-
term memory deciency. Increased step-through latencies and
decreased shuttle avoidance responses suggested hyperactivity,
decreased exploratory behavior, and impairment in learning and
memory. The impairments in this study may be attributed to the
increased hyperactivity in rats [31]. These alterations in behavior
may 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 lead
toxicity suggested that some of the effects of lead may be due
to its interference with calcium-mediated cellular processes, the
activation of protein kinases or the release of neurotransmitters
(including GABAergic and glutamatergic neurotransmission) [34].
After Pb exposure, synthesis of the inhibitory transmitter GABA is
diminished [35]. PR contains a large amount of GABA, which plays
a crucial role in memory processes. In this study, the GABA lev-
els in the hippocampi were signicantly increased after feeding
with the BR and PR diets. Therefore, the large amount of GABA
in PR may regulate the glutamatergic system by enhancing glu-
tamate release and/or the sensitivity of NMDA receptors, resulting
in 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 oxidative
stress by inducing the generation of ROS. Excess production of ROS
in the brain has been implicated as a common underlying fac-
tor for the etiology of lead-induced neurotoxicity [37,38]. Hence,
490 R. Zhang et al. / Chemico-Biological Interactions 184 (2010) 484491
combinational anti-oxidant therapies are warranted. Indirect in
vivo evidence of oxidative involvement in lead-induced pathotox-
icity was demonstrated by alleviation of oxidative stress in the
erythrocytes after treatment withthe proventhiol-containing anti-
oxidants, N-acetyl cysteine, and a succimer in lead-exposed rats
[39]. Also, ROS-related lead toxicity in rat sperm was prevented by
thesupplementationof rat fedwithvitaminEand/or vitaminC[40].
PR and BR contain abundant phenolic compounds with benecial
effects that have been attributed mainly to their anti-oxidant activ-
ity [41,42]. Inthis study, we founda signicant decline of ROS levels
in hippocampal cells in rats fed with a PR or BR diet. These recent
ndings suggest a potential role of PR and BR in the amelioration
of lead toxicity [43].
Lead could also interact with biological membranes, induc-
ing LPO [44]. Finally, this metal could decrease the activities of
free-radical scavenging enzymes, such as catalase (CAT), SOD and
GSH-Px [45]. The latter is mainly attributed to the high afnity of
Pb for sulfhydryl groups or metal cofactors in these enzymes and
molecules. Previous study indicated that the changes of SOD and
GSH-Px activities in serumand hippocampus could reect the neu-
ronal damageor evenneuronal death[46]. AndMDAlevels inserum
could serve as the biomarkers of neuropathy [47]. In this study, the
anti-oxidant enzyme activities in sera and hippocampi were mea-
sured. We found the GSH-Px and SOD activities in sera of rats in
PbAC treatment groups that were fed with the different rice diets
were signicantly increased. In hippocampi, the SOD activities of
Pb-exposed rats on BR and PR diets were signicantly increased
compared with that of the PbAC group. Our results suggested that
feeding with the PR and BR diets could improve the anti-oxidative
enzyme activities, which are the major mechanisms for reducing
local levels of ROS such as the superoxide anion radical (O
2

) and
hydrogen peroxide (H
2
O
2
) [48]. The degree of oxidative stress can
also be evaluated by determining levels of MDA in lead-exposed
rats on the different diets. A 7.9-fold elevation in serum and a 1.3-
fold elevation in hippocampal MDA levels indicated the presence
of lead-induced LPO. Feeding rice diets following lead exposure
reduced the MDA concentrations, and feeding a PR diet reduced
the MDA levels back to normal levels in serum.
Pb-induced oxidative stress damage could result from the inhi-
bition of ALAD (d-aminolevulinic acid dehydratase), leading to the
accumulation of ALA [49]. ALA in blood might be the best indicator
of the differential lead exposure over baseline levels [50]. In this
study, we found that feeding PR and BR diets could prevent the
accumulation of ALA. These changes in ALA levels in rats on dif-
ferent diets were due to the changes in Pb concentration in blood.
ALA is a potential endogenous source of free radicals and has been
shown to cause oxidative damage to DNAin Chinese hamster ovary
cells in vitro through the formation of 8-OHdG adducts [51]. The
effect of oxidative damage on DNA in humans from lead toxicity
is also supported by a recent study of 7,8-dihydro-9-oxoguanine
adducts in lymphocytes collected from persons environmentally
exposed to metals, including lead [52]. In this study, lead acetate
induced a 5.2-fold increase in the hippocampal 8-OHdG/10
5
2-
dG ratio in rats on the AIN-93G diet. Additionally, the hippocampal
8-OHdG/10
5
2-dG ratio induced 4.4-, 4.5- and 2.1-fold increases
in rats fed with the different rice diets compared with the control.
These data suggested that feeding with rice diets might prevent the
Pb-induced 8-OHdG adducts. The assumption that oxidative stress
is a mechanismthat leads to lead neurotoxicity suggests that some
anti-oxidants, including vitamins and other minerals as well as the
rice diet, could ameliorate lead neurotoxicity [43,53,54].
In this study, we found that the protective effects of PR and BR
diet on lead-exposed rats were related to their anti-oxidant activ-
ities or to the large amount of GABA present. Moreover, the levels
of Pb in the sera and hippocampi decreased in rats on the PR and
BR diets, indicating that the BR and PR diets might prevent the Pb
accumulation to some degree. It is well known that dietary ber,
vitamin E, and vitamin C are abundant in PR. Cellulose supplemen-
tationreducedthe retentionof Pbinrats [55], anddietaryber from
bran can effectively bind Pb ions, preventing toxicity [56]. There-
fore, thePRdiet might potentiallybetherapeutic inthetreatment of
chronic Pb intoxication. However, the detailed mechanismrespon-
sible for the falling Pb levels in rats fed with the PR diet requires
additional investigation.
In conclusion, we have shown that the PR diet improved learn-
ing and memory decits induced by low-level lead in young rats. In
particular, we concluded that, therapeutically, PRmay prevent lead
neurotoxicity. However, it is unclear whether the neuronal dam-
age induced can be reversed. Several mechanisms may contribute
to the apparent positive effects of PR on learning and memory.
For instance: (1) the benecial effects on lead neurotoxicity from
the abundant phenolic compounds have been mainly attributed to
their associated anti-oxidant activities such as reducing the ROS
and MDA levels, as well as the formation of 8-OHdG adducts in the
hippocampus 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 some
degree. Future studies on chronically low levels of lead exposure
and the effect of the PR diet may perhaps be able to answer many
unanswered questions. The PR diet may be a potential therapy in
the treatment of chronic Pb intoxication, primarily during devel-
opment.
Conict of interest statement
The authors declare that there are no conicts of interest.
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