ORIGINAL PAPER

Subchronic Oral Administration of Benzo[a]pyrene ImpairsMotor and Cognitive Behavior and Modulates S100B Levelsand MAPKs in Rats

Erica Santos Maciel • Regina Biasibetti • Ana Paula Costa • Paula Lunardi •

Rebeca Vargas Antunes Schunck • Gabriela Curbeti Becker • Marcelo Dutra Arbo •

Eliane Dallegrave • Carlos Alberto Goncalves • Paulo H. Nascimento Saldiva •

Solange Cristina Garcia • Rodrigo Bainy Leal • Mirna Bainy Leal

Received: 18 December 2013 / Revised: 14 February 2014 / Accepted: 15 February 2014 / Published online: 2 March 2014

� Springer Science+Business Media New York 2014

Abstract Benzo[a]pyrene (BaP) is an environmental con-

taminant produced during incomplete combustion of organic

material that is well known as a mutagenic and carcinogenic

toxin. There are few studies addressing the molecular and

cellular basis of behavioural alterations related to BaP

exposure. The aim of this study was to evaluate the effect of

subchronic oral administration of BaP on behavioral and

neurochemical parameters. Wistar male rats received BaP

(2 mg/kg) or corn oil (control), once a day for 28 days

(n = 12/group). Spontaneous locomotor activity and short-

and long-term memories were evaluated. Glial fibrillary acid

protein and S100B content in the hippocampus, serum and

CSF were measured using ELISA and total and

phosphorylated forms of mitogen activated protein kinases

(MAPKs) named extracellular signal-regulated kinases 1 and

2, p38MAPK and c-Jun amino-terminal kinases 1 and 2, in the

hippocampus, were evaluated by western blotting. BaP

induced a significant increase on locomotor activity and a

decrease in short-term memory. S100B content was increased

significantly in cerebrospinal fluid. BaP induced a decrease on

ERK2 phosphorylation in the hippocampus. Thus, BaP sub-

chronic treatment induces an astroglial response and impairs

both motor and cognitive behavior, with parallel inhibition of

ERK2, a signaling enzyme involved in the hippocampal

neuroplasticity. All these effects suggest that BaP neurotox-

icity is a concern for environmental pollution.

E. S. Maciel � R. V. A. Schunck � M. B. Leal

Programa de Pos Graduacao em Ciencias

Biologicas - Neurociencias, Instituto de Ciencias Basicas da

Saude, Universidade Federal do Rio Grande do Sul, Rua

Sarmento Leite, 500/107, Porto Alegre, RS 90050-170, Brazil

R. Biasibetti � P. Lunardi � C. A. Goncalves

Departamento de Bioquımica, Instituto de Ciencias Basicas da

Saude, Universidade Federal do Rio Grande do Sul, Rua Ramiro

Barcelos, 2600-Anexo, Santana, Porto Alegre, RS 90035-003,

Brazil

A. P. Costa � R. B. Leal

Departamento de Bioquımica, Centro de Ciencias Biologicas,

Universidade Federal de Santa Catarina, CCB - Bloco C – 28Andar – Sala 210, Campus Universitario, Florianopolis,

SC 88040-900, Brazil

G. C. Becker � M. B. Leal

Laboratorio de Farmacologia e Toxicologia de Produtos

Naturais, Departamento de Farmacologia, Instituto de Ciencias

Basicas da Saude, Universidade Federal do Rio Grande do Sul,

Rua Sarmento Leite, 500/202, Porto Alegre, RS 90050-170,

Brazil

M. D. Arbo

REQUIMTE, Laboratorio de Toxicologia, Departamento de

Ciencias Biologicas, Faculdade de Farmacia, Universidade do

Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal

E. Dallegrave

Departamento de Ciencias Basicas da Saude, Universidade

Federal de Ciencias da Saude de Porto Alegre, Rua Sarmento

Leite, 245, Porto Alegre, RS 90050-170, Brazil

P. H. N. Saldiva

Departamento de Patologia, Faculdade de Medicina,

Universidade de Sao Paulo, Av. Dr. Arnaldo, 455 - 18 andar, sala

1103, Sao Paulo, SP 01246-000, Brazil

S. C. Garcia

Laboratorio de Toxicologia, Departamento de Analises,

Faculdade de Farmacia, Universidade Federal do Rio Grande do

Sul, Av. Ipiranga, 2752, Porto Alegre, RS 90610-000, Brazil

M. B. Leal (&)

Departamento de Farmacologia, ICBS – UFRGS, Sarmento

Leite 500 sala 202, Porto Alegre, RS 90050-170, Brazil

e-mail: mirnabl@gmail.com

123

Neurochem Res (2014) 39:731–740

DOI 10.1007/s11064-014-1261-y

Keywords Benzo[a]pyrene � Locomotor activity � Object

recognition � GFAP � S100B � MAPKs

Introduction

Benzo[a]pyrene (BaP) is a member of the group of

polycyclic aromatic hydrocarbons (PAHs) which is a

widespread environmental contaminant formed during

incomplete combustion or pyrolysis of organic material.

This substance is found as a contaminant of air, water,

soils and sediments. Human exposure to significant

amounts of BaP occurs through the ingestion of con-

taminated food and water or the inhalation of particulates

in the air [1]. Automobile exhaust (especially from diesel

engines), industrial emissions and forest fires are

important sources of environmental BaP [2, 3]. Addi-

tionally, tobacco smoke contains high concentrations of

PAHs, especially BaP [3]. Several studies suggest that

BaP can cause damages to human health, being associ-

ated with pulmonary and liver cancers, cardiovascular,

pulmonary and neurodegenerative diseases and it has

been implicated in infertility and growth retardation in

childhood [4]. The role of BaP as a potent mutagen and

carcinogenic agent is well defined [5]; however, only

recently the neurotoxic effects of BaP have been

receiving attention. As a highly lipophilic compound,

BaP readily crosses the blood–brain barrier and reaches

the central nervous system (CNS) causing neurological

alterations [6, 7].

Several studies demonstrate the pivotal role of the hip-

pocampus to memory formation and learning [8, 9]. It was

noted that subchronic administration of BaP in rats preju-

dices spatial learning and short-term memory evaluated by

Morris water maze test and induces changes of hippo-

campal neurotransmitter systems [10–13] and damage of

hippocampal neurons [14], implicating BaP as a neurotoxic

pollutant that may act on the hippocampus and impair

learning and memory. In humans, occupational exposure to

BaP is associated with neurobehavioral function impair-

ment in coke oven workers [15].

Astrocytes are intimately associated with neurons and

play an important role in brain homeostasis. Glial activa-

tion in response to injury stimuli commonly involves

changes in glial fibrillary acid protein (GFAP), S100B

protein and antioxidant defenses [16, 17]. GFAP is a spe-

cific astrocyte marker; currently, tissue GFAP increase is

taken as a sign of astrogliosis, associated with conditions of

brain injury [16].

The calcium binding protein S100B is found in brain

tissue, predominantly in the astrocytes, which secrete this

molecule. Extracellular S100B plays a trophic role in

neuronal and glial cells, but elevated extracellular levels of

this protein can induce apoptosis in neural cells [18].

Elevations of cerebrospinal fluid and serum S100B levels

have been used as markers of brain insult [19, 20].

Mitogen-activated protein kinases (MAPKs) are a fam-

ily of serine-threonine kinases that amplify and integrate

signals originated from a variety of extracellular stimuli

and may regulate cell differentiation, survival, cell death

and synaptic plasticity [21, 22]. MAPKs include the

extracellular signal-regulated kinases 1 and 2 (ERK1/2),

c-Jun amino-terminal kinases 1–3 (JNK1/2/3) and

p38MAPK (a, b, c and d) [23]. All these kinases become

functional upon phosphorylation at both a threonine and a

tyrosine residue by an upstream MAPK [22, 24]. In the

hippocampus, activation of MAPK pathway may occur in

response to glutamate, through activation of NMDA and

AMPA receptors [25, 26], oxidative stress [27], growth

factors and cytokines [28] or by activation of other protein

kinases. MAPKs misregulation are associated, in many

cases, with detrimental processes including dendritic spine

loss, cell death and impairment of learning and memory

[29]. Therefore, alteration of ERK, JNK and p38MAPK

pathways by neurotoxins may imply in important func-

tional changes of CNS.

Despite numerous studies evaluating the possible

mechanisms involved in the behavioral deficits associated

with neurotoxicity induced by BaP, some parameters have

not been fully elucidated. Therefore, the aim of this study

was to evaluate the impact of oral administration of BaP on

memory and locomotion in rats, as well as the MAPK

signaling (ERK1/2, p38MAPK and JNK1/2) and astrocyte

markers in hippocampus (GFAP and S100B). Moreover,

cerebrospinal and serum S100B contents were also evalu-

ated after BaP exposure.

Materials and Methods

Chemicals

Benzo[a]pyrene (99 % purity), primary antibodies anti-

JNK1/2, anti-ERK1/2 and anti-p38MAPK, ammonium per-

sulfate (APS), o-phenylenediamine (OPD) and anti-S100B

monoclonal antibody were obtained from Sigma (St. Louis,

MO, USA). Anti-S100 and anti-GFAP antibodies were

from Dako (Carpinteria, CA, USA). Peroxidase-conjugated

secondary antibody was from Amersham International

(UK). Triton X-100, sodium dodecyl sulfate (SDS),

acrylamide and bis-acrylamide were obtained from GE

Healthcare Life Science (UK). Glycine, Tris, TEMED, bmercaptoethanol and protease inhibitor cocktail (M222)

were obtained from Amresco Life Science (Solon, OH,

USA). Bovine serum albumin (BSA) was from Inlab (Sao

732 Neurochem Res (2014) 39:731–740

123

Paulo, Brazil). Mouse anti-b actin was obtained from Santa

Cruz Biotechnology (Santa Cruz, CA, USA). Immobilon

nitrocellulose and goat anti-mouse IgG HRP (horseradish

peroxidase) was from Millipore (Billerica, MA, USA). The

anti-phospho-ERK1/2, anti–phospho-JNK1/2, anti-phos-

pho-P38MAPK antibodies and LumiGLO reagent (luminol

chemiluminescent substrate) were purchased from Cell

Signaling (Beverly, MA, USA). All other reagents were of

analytical grade.

Animals

Twenty-four male Wistar rats, adult (90 days) weighing

250–300 g, from the Center for Reproduction and Animal

Experimentation Laboratory of the Universidade Federal

do Rio Grande do Sul (UFRGS-CREAL) were used. The

experiments were performed after approval of the protocol

by the University Ethics Committee (Number 18287) and

were carried out in accordance with current guidelines for

the care of laboratory animals [30]. The animals were kept

in polypropylene cages (41 9 34 9 16 cm) (4 rats per

cage) with free access to food and water, under standard

conditions (12 h light/12 h dark cycle, at a constant tem-

perature of 22 ± 2 �C and controlled humidity).

Subchronic Administration

The experimental protocol was based on OECD (Organi-

sation for Economic Co-operation and Development)

guideline 407 (Repeated Dose 28-day Oral Toxicity Study

in Rodents) [31]. Rats were divided into 2 groups: control

(n = 12) and BaP (n = 12). BaP was dissolved in corn oil.

Animals (250–300 g) were treated by gavage for 28 con-

secutive days with corn oil (vehicle, Cargill Agricola S.A./

SP, Brazil) or BaP 2 mg/kg. This dose is equivalent to

contamination levels in smokers, consumers of high

amounts of products as smoked or grilled meat and fish, or

even individuals with high occupational exposure [1]. The

treatments were administered everyday at the same time,

and animals were acclimated 1 h in the lab before the

treatments. In the days of behavioral tests, these were

performed all in the morning. BaP was administered at a

10 ml/kg volume.

Relative Body Weight

Body weight was measured every day and the animals were

observed daily for signs of toxicity, morbidity and mor-

tality. The calculation of the relative weight was obtained

daily with the body weight of the rats, using the following

equation: relative weight = (weight of the animal 9 100)/

weight of the animal on day 1.

Measurement of Spontaneous Locomotor Activity

The method used for evaluation of spontaneous locomotor

activity was adapted from Creese et al. [32]. The animals

were placed individually in locomotor activity cages

(50 9 48 9 50 cm) equipped with six bars, each with 16

infrared light sensors that detect the relative position of the

animal on the box (Insight Equipment Ltd., Sao Paulo,

Brazil). The total distance traveled by the animal was

evaluated for 15 min (5 initial minutes considered explor-

atory activity and the 10 final minutes the test session). The

test was performed after 26 days of treatment and the

activity was performed in a dark room with no noise.

Novel Object Recognition Memory

Twenty-four hours after locomotor activity measurement,

animals were trained and tested in a novel object recognition

task as previously described [33–35]. This task is entirely

based on the spontaneous behavior and it did not involve

primary reinforcement such as food or electric shocks, thus it

could be comparable to recognition tests widely used in

subhuman primates and to memory tests currently used in

man. When animals are exposed to a familiar and a novel

object, they approach frequently and spend more time

exploring the novel object [36]. It has been used to evaluate

short- and long-term memory in rodents by several studies

[37, 38]. Training in the object recognition task took place in

the same cages used for the locomotor activity. These cages

were thus used as a context habituation trial for the recog-

nition memory task. The object recognition test required that

the rats recall which of two plastic objects they had been

previously familiarized with. Twenty-four hours after arena

exploration, training was conducted by placing individual

rats into the cages, in which two identical objects (objects A1

and A2; Duplo Lego toys) were positioned in two adjacent

corners, 9 cm from the walls. Animals were left to explore

the objects for 5 min. In a short-term memory (STM) test

given 1.5 h after training, the rats explored the locomotor

activity cages for 5 min in the presence of one familiar

(A) and one novel (B) object. In a long-term memory (LTM)

test given 24 h after training, the same rats explored the

cages for 5 min in the presence of familiar object A and a

novel object C. Recognition memory was evaluated as for

the STM test. All objects presented similar textures, colors,

and sizes, but distinctive shapes. A recognition index cal-

culated for each animal was expressed by the ratio TN/

(TF ? TN) [TF = time spent exploring the familiar object

A; TN = time spent exploring the novel object B. Between

trials the objects were washed with 10 % ethanol solution.

Exploration was defined as sniffing or touching the object

with the nose and/or forepaws. Sitting on the object was not

considered exploration.

Neurochem Res (2014) 39:731–740 733

123

Cerebrospinal Fluid and Serum Samples

At the end of the behavioural experiment, animals were

anesthetized using ketamine/xylazine (10 and 50 mg/kg,

respectively) and then positioned in a stereotaxic holder for

cerebrospinal fluid collection from the cisterna magna. The

puncture was performed using an insulin syringe (27 gauge

9 1/200 length). Rats were then removed from the stereo-

taxic apparatus and placed in a flat place; whole blood from

the caudal vena cava was collected. Serum was separated

by centrifugation at 3,0009g for 5 min. CSF and serum

samples were frozen (-20 �C) until further analysis [39].

Hippocampal Tissue Samples

The animals were killed by piercing the diaphragm, brains

were removed, and hippocampi were dissected. Right

hippocampi were used for MAPKs analysis and dissected

(4 �C) into ‘‘cutting solution’’ (110 mM sucrose, 60 mM

NaCl, 3 mM KCl, 1.25 mM KH2PO4, 0.5 mM CaCl2,

7 mM Mg2SO4, 5 mM glucose; 25 mM HEPES, pH 7.4)

and placed in liquid nitrogen for storage at -80 �C until

use [40]. Left hippocampi were placed in cold saline

medium with the following composition: 120 mM NaCl;

2 mM KCl; 1 mM CaCl2; 1 mM MgSO4; 25 mM HEPES;

1 mM KH2PO4 and 10 mM glucose, adjusted to pH 7.4 and

used for GFAP and S100B quantification.

ELISA for S100B and GFAP

The S100B content in the CSF and serum was measured by

ELISA [41]. Briefly, 50 ll of sample plus 50 ll of Tris

buffer were incubated for 2 h on a microtiter plate previ-

ously coated with monoclonal anti-S100B (SH-B1). Poly-

clonal anti-S100B was incubated for 30 min and then

peroxidase-conjugated anti-rabbit antibody was added for a

further 30 min. A colorimetric reaction with o-phenylene-

diamine was measured at 492 nm. The standard S100B

curve ranged from 0.025 to 2.5 ng/mL. ELISA for GFAP

in the hippocampus [42] was carried out by coating the

microtiter plate with 100 ll samples containing 30 lg of

protein for 48 h at 4 �C. Incubation with a rabbit poly-

clonal anti-GFAP for 2 h was followed by incubation with

a secondary antibody conjugated with peroxidase for 1 h,

at room temperature; the standard GFAP curve ranged

from 0.1 to 10 ng/mL.

Western Blotting for MAPKs

The samples were prepared as previously described [43].

Briefly, the samples were mechanically homogenized in

400 ll in 50 mM Tris, pH 7.0, 1 mM EDTA, 100 mM

NaF, 0.1 mM PMSF, 2 mM Na3VO4, 1 % Triton X-100,

10 % glycerol and protease inhibitor cocktail. Thereafter,

the samples were incubated on ice for 10 min. Lysates

were centrifuged (10,0009g at 4 �C for 10 min) to elimi-

nate cellular debris. The supernatants were diluted 1/1 (v/v)

in 100 mM Tris, pH 6.8, 4 mM EDTA, and 8 % SDS and

were then boiled for 5 min. Subsequently, the loading

buffer (40 % glycerol, 100 mM Tris, bromophenol blue,

pH 6.8) in the ratio 25:100 (v/v) and b-mercaptoethanol

(final concentration 8 %) were added to the samples. Pro-

tein content was estimated by the method described by

Peterson [44]. The same amount of protein (70 lg per lane)

for each sample was electrophoresed in 10 % sodium

dodecyl sulfate polyacrylamide gel electrophoresis

(SDS–PAGE) minigels [45, 46] and transferred to nitro-

cellulose membranes using a semidry blotting apparatus

(1.2 mA/cm2; 1.5 h) as described by Bjerrum and Heeg-

aard [47]. To verify the transfer efficiency process, the gels

Fig. 1 Effects of BaP on relative weight variation of rats.

Benzo[a]pirene 2 mg/kg (BaP) or vehicle (corn oil) were adminis-

tered orally (by gavage) once a day during 28 days. Relative

weight = (weight of the animal 9 100)/weight of the animal on

day 1. Values represent mean ± SEM (n = 12, one-way repeated

measurements ANOVA, p \ 0.05)

Fig. 2 Effects of BaP on spontaneous locomotor activity of rats.

Benzo[a]pyrene 2 mg/kg (BaP) or corn oil (control) administered

orally (by gavage) once a day during 28 days. Each column represents

the mean ± SEM. (n = 12 per group). *p \ 0.05 versus vehicle—

treated group by Student’s t test

734 Neurochem Res (2014) 39:731–740

123

were stained with Coomassie blue and the membranes were

stained with Ponceau S. The membranes were blocked for

1 h with 5 % skim milk in Tris-buffered saline (TBS;

10 mM Tris, 150 mM NaCl, pH 7.5). The proteins were

detected using specific antibodies diluted in TBS-T con-

taining BSA (2 %) in the ratio: 1:5,000 for anti-phospho

ERK1/2; 1:1,000 for both anti-phospho P38MAPK, anti-

phospho JNK1/2/3 and anti-total JNK1/2; 1:40,000 for

anti-total-ERK1/2; 1:10,000 for anti-total p38MAPK;

1:2,000 for b-actin. The reactions were developed by

chemiluminescence (LumiGLO�). All steps of blocking

and incubation were followed by three times washing

(5 min) of the membranes with TBS-T (Tris 10 mM, NaCl

150 mM, Tween-20 0.05 %, pH 7.5).

Quantification and Statistical Analysis

The relative body weight was analyzed by one-way repe-

ated measures ANOVA. Comparisons between groups in

novel object recognition memory test were performed

using Mann–Whitney U test. Locomotor activity, S100B

(CSF, serum and hippocampus) and hippocampal GFAP

were analyzed by Student’s t test.

The optical density (O.D.) of the bands was quantified

using Scion Image� software. The phosphorylation level

was determined as a ratio of O.D of the phosphorylated

band/O.D. of the total band [48]. Data are expressed as

percentage of the control (considered as 100 %) and the

values are presented as mean ± S.E.M. Statistical signifi-

cance was assessed by Student’s t test. A value of p \ 0.05

was considered significant.

Results

Effect of Subchronic BaP Administration on Body

Weight

Data of the relative body weights (0, 7, 14, 21 and 28 days)

are show in Fig. 1. There was no significant difference

between vehicle and BaP-treated animals.

Effect of Subchronic BaP Administration on Motor

Behavior

Figure 2 shows the effects of BaP on total distance traveled by

animals. The total distance in group treated with BaP 2 mg/kg

(842.86 ± 94.71 cm) has increased significantly (p \ 0.021)

in comparison to control group (551.79 ± 67.39 cm) after

26 days of treatment.

Fig. 3 Effect of BaP on recognition memory deficits of rats.

Benzo[a]pyrene 2 mg/kg (BaP) or vehicle (corn oil) were administered

orally (by gavage) once a day during 28 days. a Training, b short-term

memory test (STM) was performed 1.5 h after training and c long-term

memory (LTM) at 24 h after training. ‘‘Recognition Index’’ expressed

by the ratio TN/TF ? TN, TF = time spent exploring the familiar

object and TN = time spent exploring the novel object. Data expressed

as median [interquartiles ranges], (n = 12). Comparisons between

groups—Mann–Whitney U test.*p \ 0.05

Neurochem Res (2014) 39:731–740 735

123

Effect of Subchronic BaP Administration on Cognitive

Behavior

Figure 3 shows the effect of BaP on the object recognition

memory task. There was no significant difference between

groups in the training trial. However, BaP-treated animals

showed a significant deficit in the short term memory. The STM

retention trial presented median [interquartile ranges] recog-

nition indices of 55.9 [52.5/58.4] in the control group and 45.7

[43.3/51.1] in the group treated with BaP (p\ 0.05). There was

no significant difference between groups in long-term memory.

Effect of Subchronic BaP Administration on Glial

Markers

Regarding the glial marker analysis, there was no signifi-

cantly differences in GFAP immunocontent in hippocam-

pus of BaP-treated rats (p = 0.07) evaluated by ELISA

(Fig. 4a). Hippocampal S100B content did not present any

alteration (Fig. 4b), however extracellular levels of this

protein changed in BaP-treated rats. S100B increased in the

CSF (p = 0.004; Fig. 4c) and decreased in the serum

(p = 0.04; Fig. 4d) of these animals.

Effect of Subchronic BaP Administration on MAPKs

Pathways

Concerning the evaluation of MAPKs, it was observed that

BaP-treated animals showed a significant decrease in the

level of phosphorylation of ERK2 (p = 0.016), but not on

ERK1 (Fig. 5a). Moreover, there were no alterations in the

phosphorylation of JNK1/2/3 (Fig. 5b), and p38MAPK

(Fig. 5c). The total content of each MAPK was not altered

by the treatment.

Discussion

In the present work we demonstrated that BaP administered

in vivo at 2 mg/kg for 28 consecutive days caused altera-

tions in locomotor activity and induced cognitive deficit.

Moreover, BaP caused changes in extracellular levels of

S100B and provoked a decrease in the hippocampal ERK2

activity, an important signaling enzyme which participates

in the synaptic plasticity process. All these effects indicate

that BaP causes neurotoxicity in a dose that does not alter

the body mass gain of the animals throughout the treatment.

Fig. 4 Effect of BaP on GFAP in the hippocampus and S100B in

hippocampus and extracellular compartments of rats. Benzo[a]pyrene

2 mg/kg (BaP) or vehicle (corn oil) were administered orally (by

gavage) once a day during 28 days. Contents of GFAP and S100B

were measured by ELISA. a GFAP content in hippocampus; b S100B

levels in hippocampus; c cerebrospinal fluid (CSF) S100B level

(collected by cisterna magna puncture) and d serum S100B level

(collected from the caudal cava vena). Values are mean ± SE

(n = 12 per group). *Significantly different between groups (Stu-

dent’s t test, p \ 0.05)

736 Neurochem Res (2014) 39:731–740

123

In the present study, it was observed an increase in

locomotor activity of animals after subchronic oral

administration of BaP (2 mg/kg). Some studies observed

decrease in the distance traveled after single dose treatment

of BaP [49, 50]. However, these studies applied higher

doses of BaP (25–200 mg/kg) and also a single adminis-

tration. In a pilot study realized by our group with a lower

dose of BaP (1 mg/kg) administered for 28 consecutive

days, it was not verified any difference in locomotor

activity (unpublished data).

In our study, BaP-treated animals showed deficits in

short term memory, however, this deficiency was not

observed in long-term memory. The best results with this

test are generally found between sixty to ninety minutes

after training [51]. The longest time between training and

testing long-term memory deteriorates the performance of

novel object recognition. The deficit of object recognition

memory is, in part, in agreement with other studies that

showed deficits in another type of memory, the spacial

memory, of the Morris water maze test [10–12, 14].

Among brain structures involved in the formation and

retention of memory, the hippocampus plays a key role.

Regarding this aspect animals treated with BaP (2 mg/kg)

for 90 days showed neuronal ultrastructural alterations that

include vacuole formation, from swollen and distorced

mitochondria, endoplasmic reticulum expansion, Golgi

apparatus disruption, nucleolus collapse or fragmentation

and myelin sheath degeneration in hippocampal neurons as

evaluated by transmission electron microscopy [14]. In our

protocol, the 28 days BaP exposure caused neurochemical

alterations in the hippocampus as well as disturbed behav-

ioral tasks dependent of hippocampus. Therefore, our study

reinforces the hippocampus as one possible target for BaP.

In our knowledge this is the first study that evaluates the

impact of BaP exposure in vivo on glial marker proteins

GFAP and S100B. In brain tissue, following injury, either

as a result of mechanical trauma, disease, or chemical

insult, astroglial cells become reactive or respond in a

b Fig. 5 Effect on MAPK’s in the hippocampus of rats submitted to

BaP. Benzo[a]pyrene 2 mg/kg (BaP) or vehicle (corn oil) were

administered orally (by gavage) once a day during 28 days.

Phosphorylation of p38MAPK, JNK1/2, ERK1/2 was detected by

specific antibodies against the biphosphorylated region of each kinase

and the reactions were developed by ECL. The panels show

representative western blot and quantitative analysis of phosphory-

lation for each MAPK. a ERK1/2; b JNK 1/2 and c p38MAPK. The

data are expressed as percentages of the control (considered as

100 %). Values are mean ± SE of 5–6 rats in each group. *Signif-

icant differences from control (Student’s t test, p \ 0.05)

Neurochem Res (2014) 39:731–740 737

123

typical manner, termed astrogliosis [16]. It has been

reported that benzo[a]pyrene diol epoxide, a metabolic

product of BaP, was able to activate NF-kappa B in rat

astrocyte cultures [51].

The response of glial cells to BaP is confirmed by

changes observed in CSF S100B. S100B is a calcium

binding protein expressed and mainly secreted by astrocytes

in vertebrate brain. Intracellularly, S100B binds to many

protein targets, possibly modulating cytoskeleton plasticity,

cell proliferation and astrocyte energy metabolism [18]. We

did not find any changes in S100B immunocontent in hip-

pocampus of BaP-treated animals. On the other hand, the

present study showed that CSF S100B is high in the BaP-

treated animals. This elevation suggests an astrocyte acti-

vation following brain injury [52–54] caused by BaP

administration. It is important consider that astrocytes

regulate endothelial cells of blood brain barrier (BBB) [55],

which in turn also are targets of BaP toxicity [56]. This

aspect must be taken in account to interpret changes in CSF

and serum S100B distribution reported here.

In serum, unlike the CSF, BaP-treated animals showed a

decrease in the levels of S100B. Serum S100B elevation is

frequently assumed as consequence of CSF S100B incre-

ment [53]. Therefore, it would be possible to conceive that

serum S100B decrease reflects CSF ‘‘retention’’ of this

protein, as consequence of BBB alteration induced by BaP.

Moreover, in agreement with many data in the literature,

our data reinforce the idea that changes in CSF S100B are

not necessarily accompanied by changes in serum S100B.

It is important to mention that many extracerebral sources

contribute to serum S100B, particularly adipocytes [57]. In

fact, many brain disorders are accompanied by changes

(increase or decrease) in serum S100B. A decrease in

serum S100B, as observed in our study, was observed in

Alzheimer’s disease patients [58]. It is possible that serum

S100B changes observed here after BaP administration is

due to effects on extracerebral sources of S100B-contain-

ing cells, independent of brain effect as observed in LPS

administration [59]. However, further studies are necessary

to confirm this possibility.

Recent studies demonstrated in vitro that BaP induces

activation of mitogen activated protein kinase family

members, such as p38MAPK and ERK, but not JNK, in

HepG2 cells and in human embryo lung fibroblasts among

other. In HL60 cells, the treatment with a metabolite of the

BaP also induced activation of ERK1/2 and p38 but not

JNK [60, 61].

This is the first study reporting the effect of BaP,

administered in vivo, on MAPKs. In our results, ERK2 was

inhibited in BaP-treated animals. Studies have indicated

ERK signalling as a crucial player in synaptic and neuronal

plasticity and also in learning and memory. In this context

it is interesting to note that there are multiple upstream

regulators of ERK in the hippocampus, such as: norepi-

nephrine, dopamine, histamine, serotonin, glutamate,

estrogen and brain derived neurotrophic factor (BDNF)

[21, 22], which could be involved in the alteration of the

enzyme activity observed in response to BaP exposure. In

spite of the unclear mechanism involved in the ERK

modulation by BaP, it is important to consider that ERK1/2

display multiples functions associated with synaptic plas-

ticity [21], cell survival and proliferation [62, 63] that

might be relevant in the molecular mechanisms involved in

BaP neurotoxicity. Regarding this point it is important to

highlight that hippocampal inhibition of ERK phosphory-

lation by MEK inhibitors (ex. U0126) impairs short term

memory formation [64]. Therefore, our findings suggest the

possibility that ERK inhibition by BaP might be part of the

molecular mechanism involved in the cognitive deficits.

However, future studies will be necessary to demonstrate

this aspect.

In conclusion, subchronic adminstration of BaP in Wi-

star rats affected locomotion and impaired cognition,

evaluated by a task dependent on hippocampus integrity.

The altered ERK signaling observed in the hippocampus of

these animals could contribute to understanding the cog-

nitive deficit. Moreover, we found an increase in the CSF

S100B levels indicating an astroglial activation induced by

BaP. However, we found only a trend of GFAP content

increment in the hippocampus. Although our data reveals

the sensitivity of hippocampus to BaP subchronic expo-

sure, further studies involving different hippocampal sub-

regions and other neurochemical parameters, are necessary

to characterize the mechanism(s) involved in the toxicity

produced by BaP in this brain region.

Acknowledgments The study was supported by the Conselho

Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq),

Coordenacao de Aperfeicoamento de Pessoal de Nıvel Superior

(CAPES) and Instituto Nacional de Ciencia e Tecnologia para (INCT)

a Analise Integrada do Risco Ambiental.

Conflict of interest The authors declared that there are no conflicts

of interest.

References

1. ATSDR (1995) Toxicological profile for polycyclic aromatic

hydrocarbons (PAHs). In: Agency for Toxic Substances and

Disease Registry, Atlanta, GA

2. Liu SH, Wang JH, Chuu JJ, Lin-Shiau SY (2002) Alterations of

motor nerve functions in animals exposed to motorcycle exhaust.

J Toxicol Environ Health A 65:803–812

3. IARC (2012) Benzo[a]pyrene. NB: overall evaluation upgraded

to Group 1 based on mechanistic and other relevant data. IARC

Monogr Eval Carcinog Risk Chem Man 92:111–144

4. Enomoto M, Tierney WJ, Nozaki K (2008) Risk of human health

by particulate matter as a source of air pollution—comparison

with tobacco smoking. J Toxicol Sci 33:251–267

738 Neurochem Res (2014) 39:731–740

123

5. IARC (1973) Certain polycyclic aromatic hydrocarbons and

heterocyclic compounds. IARC Monogr Eval Carcinog Risk

Chem Man 3:1–271

6. Das M, Mukhtar H, Seth P (1985) Distribution of benzo(a)pyrene

in discrete regions of rat brain. Bull Environ Contam Toxicol

35:500–504

7. Yan T, Chengzhi C, Haiyan Y, Baijie T (2010) Distribution of

benzo[a]pyrene in discrete regions of rat brain tissue using light

microscopic autoradiography and gamma counting. Toxicol

Environ Chem 92:1309–1317

8. Zola-Morgan SM, Squire LR (1990) The primate hippocampal

formation: evidence for a time-limited role in memory storage.

Science 250:288–290

9. Lubenov EV, Siapas AG (2009) Hippocampal theta oscillations

are travelling waves. Nature 459:534–539

10. Tang Q, Xia YY, Cheng SQ, Tu BJ (2011) Modulation of

behavior and glutamate receptor mRNA expression in rats after

sub-chronic administration of benzo(a)pyrene. Biomed Environ

Sci 24:408–414

11. Xia Y, Cheng S, He J, Liu X, Tang Y, Yuan H, He L, Lu T, Tu B,

Wang Y (2011) Effects of subchronic exposure to benzo[a]pyrene

(B[a]P) on learning and memory, and neurotransmitters in male

Sprague–Dawley rat. Neurotoxicology 32:188–198

12. Nie J, Duan L, Yan Z, Niu Q (2013) Tau hyperphosphorylation is

associated with spatial learning and memory after exposure to

benzo[a]pyrene in SD rats. Neurotox Res 24:461–471

13. Patri M, Singh A, Mallick BN (2013) Protective role of nor-

adrenaline in benzo[a]pyrene-induced learning impairment in

developing rat. J Neurosci Res 91:1450–1462

14. Chengzi C, Yan T, Shuqun C, Xuejun J, Youbin Q, Yinyin X,

Quian T, Baijie T (2011) New candidate proteins for benzo(a)-

pyrene-induced spatial learning and memory deficits. J Toxicol

Sci 36:163–171

15. Chongying Q, Bin P, Shuqun C, Yinyian X, Baijie T (2013) The

effect of occupational exposure to benzo[a]pyrene on neurobe-

havioral function in coke oven workers. Am J Ind Med

56:347–355

16. Eng LF, Ghirnikar RS, Lee YL (2000) Glial fibrillary acidic

protein: GFAP-thirty-one years (1969–2000). Neurochem Res

25:1439–1451

17. Donato R, Sorci G, Riuzzi F, Arcuri C, Bianchi R, Brozzi F,

Tubaro C, Giambanco I (2009) S100B’s double life: intracellular

regulator and extracellular signal. Biochim Biophys Acta

1793:1008–1022

18. Van Eldik LJ, Wainwright MS (2003) The Janus face of glial-

derived S100B: beneficial and detrimental functions in the brain.

Restor Neurol Neurosci 21:97–108

19. Andreazza AC, Cassini C, Rosa AR, Leite MC, de Almeida LM,

Nardin P, Cunha AB, Cereser KM, Santin A, Gottfried C, Sal-

vador M, Kapczinski F, Goncalves CA (2007) Serum S100B and

antioxidant enzymes in bipolar patients. J Psychiatr Res

41:523–529

20. Vicente E, Boer M, Leite M, Silva M, Tramontina F, Porciuncula

L, Dalmaz C, Goncalves CA (2004) Cerebrospinal fluid S100B

increases reversibly in neonates of methyl mercury-intoxicated

pregnant rats. Neurotoxicology 25:771–777

21. Sweatt JD (2004) Mitogen-activated protein kinases in synaptic

plasticity and memory. Curr Opin Neurobiol 14:1–7

22. Thomas GM, Huganir RL (2004) MAPK cascade signalling and

synaptic plasticity. Nat Rev Neurosci 5:173–183

23. Cargnello M, Roux PP (2011) Activation and function of the

MAPKs and their substrates, the MAPK-activated protein kina-

ses. Microbiol Mol Biol Rev 75:50–83

24. Chen Z, Gibson TB, Robinson F, Silvestro L, Pearson G, Xu B,

Wright A, Vanderbilt C, Cobb MH (2001) MAP kinases. Chem

Rev 101:2449–2476

25. Jerusalinsky D, Ferreira MB, Walz R, Da Silva RC, Bianchin M,

Ruschel AC, Zanatta MS, Medina JH, Izquierdo I (1992)

Amnesia by post-training infusion of glutamate receptor antag-

onists into the amygdala, hippocampus, and entorhinal cortex.

Behav Neural Biol 58:76–80

26. Sweatt JD (2001) The neuronal MAP kinase cascade: a bio-

chemical signal integration system subserving synaptic plasticity

and memory. J Neurochem 76:1–10

27. Runchel C, Matsuzawa A, Ichijo H (2011) Mitogen-activated

protein kinases in mammalian oxidative stress responses. Anti-

oxid Redox Signal 15:205–218

28. Kim EK, Choi EJ (2010) Pathological roles of MAPK signaling

pathways in human diseases. Biochim Biophys Acta 1802:396–405

29. Sanderson JL, Dell’Acqua ML (2011) AKAP signaling com-

plexes in regulation of excitatory synaptic plasticity. Neurosci-

entist 17:321–336

30. Olfert ED, Cross BM, McWilliam A (1998) Manual sobre el

cuidado y uso de los animales de experimentacion. Canadian

Council on Animal Care, Ontario

31. OECD (1995) Organisation for Economic Co-operation and

Development. Guideline 407: repeated Dose 28-day Oral Tox-

icity Study in Rodent. Head of Publications Service, Paris

32. Creese I, Burt DR, Snyder SH (1976) Dopamine-receptor binding

predicts clinical and pharmacological potencies of antischizo-

phrenic drugs. Science 192:481–483

33. Schroder N, O’Dell SJ, Marshall JF (2003) Neurotoxic meth-

amphetamine regimen severely impairs recognition memory in

rats. Synapse 49:89–96

34. de Lima MNM, Laranja DC, Caldana F, Bromberg E, Roesler R,

Schroder N (2005) Reversal of age-related deficits in object

recognition memory in rats with l-deprenyl. Exp Gerontol

40:506–511

35. de Lima MNM, Polydoro M, Laranja DC, Bonatto F, Bromberg

E, Moreira JC, Dal-Pizzol F, Schroder N (2005) Recognition

memory impairment and brain oxidative stress induced by post-

natal iron administration. Eur J Neurosci 2:2521–2528

36. Ennaceur A, Delacour J (1988) A new one-trial test for neuro-

biological studies of memory in rats. 1. Behavioral data. Behav

Brain Res 31:47–59

37. Soellner DE, Grandys T, Nunez JL (2009) Chronic prenatal

caffeine exposure impairs novel object recognition and radial arm

maze behaviors in adult rats. Behav Brain Res 205:191–199

38. Moojen VKM, Damiani-Neves M, Bavaresco BV, Pescador BB,

Comim CM, Quevedo J, Boeck CR (2012) NMDA precondi-

tioning prevents object recognition memory impairment and

increases brain viability in mice exposed to traumatic brain

injury. Brain Res 23:82–90

39. Netto CB, Conte S, Leite MC, Pires C, Martins TL, Vidal P,

Benfato MS, Giugliani R, Goncalves CA (2006) Serum S100B

protein is increased in fasting rats. Arch Med Res 37:683–686

40. Lopes MW, Soares FM, de Mello N, Nunes JC, Caiado AG, de

Brito D, de Cordova FM, da Cunha RM, Walz R, Leal RB (2013)

Time-dependent modulation of AMPA receptor phosphorylation

and mRNA expression of NMDA receptors and glial glutamate

transporters in the rat hippocampus and cerebral cortex in a

pilocarpine model of apilepsy. Exp Brain Res 226:153–163

41. Leite MC, Galland F, Brolese G, Guerra MC, Bortolotto JW,

Freitas R, Almeida LM, Gottfried C, Goncalves CA (2008) A

simple, sensitive and widely applicable ELISA for S100B:

methodological features of the measurement of this glial protein.

J Neurosci Methods 169:93–99

42. Tramontina F, Leite MC, Cereser K, de Souza DF, Tramontina

AC, Nardin P, Andreazza AC, Gottfried C, Kapczinski F, Gon-

calves CA (2007) Immunoassay for glial fibrillary acidic protein:

antigen recognition is affected by its phosphorylation state.

J Neurosci Methods 162:282–286

Neurochem Res (2014) 39:731–740 739

123

43. Oliveira CS, Rigon AP, Leal RB, Rossi FM (2008) The activation

of ERK1/2 and p38 mitogen-activated protein kinases is

dynamically regulated in the developing rat visual system. Int J

Dev Neurosci 26:355–362

44. Peterson GLA et al (1977) A simplification of the protein assay

method of Lowry et al. which is more generally applicable. Anal

Biochem 83:346–356

45. Leal RB, Cordova FM, Herd L, Bobrovskaya L, Dunkley PR

(2002) Lead-stimulated p38 MAPK-dependent Hsp27 phosphor-

ylation. Toxicol Appl Pharmacol 178:44–51

46. Cordova FM, Rodrigues AL, Giacomelli MB, Oliveira CS, Posser

T, Dunkley PR, Leal RB (2004) Lead stimulates ERK1/2 and p38

MAPK phosphorylation in the hippocampus of immature rats.

Brain Res 998:65–72

47. Bjerrum OJH, Heegaard NHH (1988) CRC handbook of immu-

noblotting of proteins, vol I. CRC Press, London

48. Posser T, de Aguiar CB, Garcez RC, Rossi FM, Oliveira CS,

Trentin AG, Neto VM, Leal RB (2007) Exposure of C6 glioma

cells to Pb(II) increases the phosphorylation of p38(MAPK) and

JNK1/2 but not of ERK1/2. Arch Toxicol 81:407–414

49. Saunders CR, Ramesh A, Shockley DC (2002) Modulation of

neurotoxic behavior in F-344 rats by temporal disposition of

benzo(a)pyrene. Toxicol Lett 129:33–45

50. Saunders CR, Das SK, Ramesh A, Shockley DC, Mukherjee S

(2006) Benzo(a)pyrene-induced acute neurotoxicity in the F-344

rat: role of oxidative stress. J Appl Toxicol 26:427–438

51. Sik A, van Nieuwehuyzen P, Prickaerts J, Blokland A (2003)

Performance of different mouse strains in an object recognition

task. Behav Brain Res 147:49–54

52. Farina M, Cereser V, Portela LV, Mendez A, Porciuncula LO,

Fornaguera J, Goncalves CA, Wofchuk ST, Rocha JB, Souza DO

(2005) Methylmercury increases S100B content in rat cerebro-

spinal fluid. Environ Toxicol Pharmacol 19:249–253

53. Goncalves CA, Leite MC, Nardin P (2008) Biological and

methodological features of the measurement of S100B, a putative

marker of brain injury. Clin Biochem 41:755–763

54. Michetti F, Corvino V, Geloso MC, Lattanzi W, Bernardini C,

Serpero L, Gazzolo D (2012) The S100B protein in biological

fluids: more than a lifelong biomarker ofbrain distress. J Neuro-

chem 120:644–659

55. Alvarez JI, Katayama T, Prat A (2013) Glial influence on the

blood brain barrier. Glia 61:1939–1958

56. Gu J, Chan LS, Wong CK, Wong NS, Wong CK, Leung KN,

Mak NK (2011) Effect of benzo[a]pyrene on the production of

vascular endothelial growth factor by human eosinophilic leu-

kemia EoL-1 cells. J Environ Pathol Toxicol Oncol 30:241–249

57. Goncalves CA, Leite MC, Guerra MC (2010) Adipocytes as an

important source of serum S100B and possible roles of this

protein in adipose tissue. Cardiovasc Psychiatry Neurol

2010:790431

58. Chaves ML, Camozzato AL, Ferreira ED, Piazenski I, Kochhann

R, Dall’Igna O, Mazzini GS, Souza DO, Portela LV (2010)

Serum levels of S100B and NSE proteins in Alzheimer’s disease

patients. J Neuroinflamm 7:6

59. Guerra MC, Tortorelli LS, Galland F, Da Re C, Negri E, Engelke

DS, Rodrigues L, Leite MC, Goncalves CA (2011) Lipopoly-

saccharide modulates astrocytic S100B secretion: a study in

cerebrospinal fluid and astrocyte cultures from rats. J Neuroin-

flamm 8:128

60. Lin T, Mak NK, Yang MS (2008) MAPK regulate p53 dependent

cell death induced by benzo[a]pyrene: involvement of p53

phosphorylation and acetylation. Toxicology 247:145–153

61. Song MK, Kim YJ, Song M, Choi HS, Park YK, Ryu JC (2011)

Polycyclic aromatic hydrocarbons induce migration in human

hepatocellular carcinoma cells (HepG2) through reactive oxygen

species-mediated p38 MAPK signal transduction. Cancer Sci

102:1636–1644

62. Torii S, Nakayama K, Yamamoto T, Nishida E (2004) Regulatory

mechanisms and function of ERK MAP kinases. J Biochem

136:557–561

63. Iryo Y, Matsuoka M, Wispriyono B, Sugiura T, Igisu H (2000)

Involvement of the extracellular signal regulated protein kinase

(ERK) pathway in the induction of apoptosis by cadmium chlo-

ride in CCRFCEM cells. Biochem Pharmacol 60:1875–1882

64. Igaz LM, Winograd M, Cammarota M, Izquierdo LA, Alonso M,

Izquierdo I, Medina JH (2006) Early activation of extracellular

signal-regulated kinase signaling pathway in the hippocampus is

required for short-term memory formation of a fear-motivated

learning. Cell Mol Neurobiol 26:989–1002

65. Weng MW, Hsiao YM, Chen CJ, Wang JP, Chen WC, Ko JL

(2004) Benzo[a]pyrene diol epoxide up-regulates COX-2

expression through NF-kappaB in rat astrocytes. Toxicol Lett

151:345–355

66. Bae MK, Kim SR, Lee HJ, Wee HJ, Yoo MA, Ock OS, Baek SY,

Kim BS, Kim JB, Sik-Yoon, Bae SK (2006) Aspirin-induced

blockade of NF-kappaB activity restrains up-regulation of glial

fibrillary acidic protein in human astroglial cells. Biochim Bio-

phys Acta 1763:282–289

67. Liberto CM, Albrecht PJ, Herx LM, Yong VW, Levison SW

(2004) Pro-regenerative properties of cytokine-activated astro-

cytes. J Neurochem 89:1092–1100

740 Neurochem Res (2014) 39:731–740

123