Correspondence: Baijie Tu (E-mail: tubaijie@163.com)

Adverse effect of sub-chronic exposure to benzo(a)pyrene and protective effect of butylated hydroxyanisole on

learning and memory ability in male Sprague-Dawley ratXiao Liang1, Yan Tang2, Li Duan1, Shuqun Cheng1, Long Luo1, Xianqing Cao3

and Baijie Tu1

1Department of Occupational and Environmental Medicine, School of Public Health, Chongqing Medical University, No.1 Yixueyuan Road, Yuzhong District, Chongqing 400016, China

2Department of Occupational and Environmental Medicine, School of Public Health, Luzhou Medical College, No.1 Xianglin Road, Longmatan District, Luzhou, Sichuan, 646000, China

3Experiment center, School of Public Health, Chongqing Medical University, No.1 Yixueyuan Road, Yuzhong District, Chongqing 400016, China

(Received April 10, 2014; Accepted July 11, 2014)

ABSTRACT — Previous studies demonstrate that benzo(a)pyrene (B(a)P) can affect hippocampal func-tion and cause spatial cognition impairment. However, the mechanism is incomplete. Some evidence implies that B(a)P may cause an oxidative damage linking to the function of the hippocampus and antioxi-dant can prevent the oxidative damage in rats, but the ATPase and Ca2+ in the hippocampus and the pro-tective effect of butylated hydroxyanisole (BHA) have not been studied. This study aimed to investigate the damage of toxicity further induced by B(a)P in hippocampus and the protective effect of BHA. Nine-ty-six male Sprague-Dawley (SD) rats were randomly divided into four groups (solvent control group, BHA-group, B(a)P-exposed group and B(a)P-BHA-combination group), with daily administration for 90 days. Morris water maze (MWM) was employed to evaluate the learning and memory ability. The levels of malonaldehyde (MDA) content, superoxide dismutase (SOD) activity, Na+-K+-ATPase and Ca2+-Mg2+-ATPase activity in hippocampus were measured by commercial kits. The concentration of Ca2+ in rat hip-pocampus was detected by fluorescent labeling. In behavior test it showed that there was an adverse effect on rats in the B(a)P -group. The levels of MDA content and Ca2+ content were significantly increased in the B(a)P group, while the activities of SOD and ATPase were significantly decreased. In the B(a)P-BHA group, the change of each index diminished significantly. The results suggested that the neurobehavioral toxicity of B(a)P might have a close relationship with oxidative damage, resulted in decreasing of ATPase activities and increasing of Ca2+ concentration in the hippocampus. Furthermore, BHA can prevent these damages.

Key words: Benzo(a)pyrene, BHA, Oxidant stress, ATPase, Ca2+

INTRODUCTION

PAHs are formed during incomplete combustion and attached to the surface of particulate matter. They are prevalent and consistently present in air, water, food and dust. In the U.S., the average person can take in 0.207 μg PAHs air, 0.027 μg from water, and 0.16-l.6 μg from food. In the soil of unpolluted areas, the content of PAHs is between 5-100 μg/kg, while in the soil of polluted areas, such as highway margins, the content of PAHs can reach a range of 2-5 mg/kg. In addition, the average content of

PAHs in food is 0.002-0.9 mg/kg (Mumtaz et al., 1996). For its chemical characteristics, benzo(a)pyrene (B(a)P) is apt to produce a toxic effect in the central nervous sys-tem by passing through the blood-brain barrier (Saunders et al., 2006). In recent years, many studies have forcused on its neurotoxicity, but research on the mechanism is incomplete (Mumtaz et al., 1996).

The Brain may be extremely susceptible to attack by reactive oxygen species derived from B(a)P and/or B(a)P metabolism due to its characteristics, such as high oxygen consumption, high iron and lipid content and low

Original Article

The Journal of Toxicological Sciences (J. Toxicol. Sci.)Vol.39, No.5, 739-748, 2014

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level of antioxidants (LeBel and Bondy, 1991). Oxida-tive stress can alter brain activity, including neurotrans-mission, and cause neuronal cell apoptosis (LeBel and Bondy, 1991; Cardozo-Pelaez et al., 1999). B(a)P-induced acute neurobehavioral toxicity through oxidative stress has been observed (Saunders et al., 2006). Alterations in the dopaminergic and serotoninergic systems throughout the brain have been reported after single administration of B(a)P (Jayasekara et al., 1992; Stephanou et al., 1998). Acetylcholinesterase (AChE) inhibition has also been found in vitro when B(a)P was administered (Jett et al., 1999). These findings support the hypothesis that PAHs can alter cellular constituents and cause tissue damage in the CNS (Andersson et al., 1998). The hippocampus is essential for the regulation of spatial learning and mem-ory processes in animals (O'Keefe, 1999). Thus, the alter-nation in the neurotransmitter systems including cholin-ergic and monoaminergic system, and oxidative stress in this region may contribute to the impairment of learning and memory.

Antioxidants (such as butylated hydroxyanisole (BHA)) are important substances, which have been used to postpone and prevent oxidative degradation process-es in food products, polymers, fats and oils. Also anti-oxidants play a role in the oxidative processes of aging in human and other animals (Tamta et al., 2006; Lin et al., 2013). Because of their physicochemical properties, these compounds are used alone or in mixtures with edi-ble oils or lipid-based food in order to prevent oxidation (Medeiros et al., 2010). Several studies show that BHA is one of the several widely used antioxidant food additives, and it has been suggested to provide protection against chemical carcinogens, and immunosuppressive and anti-inflammatory functions.

Na+-K+-ATPase and Ca2+-Mg2+-ATPase are crucial enzymes in the course of energy metabolism. They have a relationship of the generation of the membrane proteins necessary to maintain neuronal excitability and cellular volume control (Erecińska and Silver, 1994). Being cru-cial for brain development and function, the concentra-tion of those two enzymes in brain cellular membranes is relatively higher than in other tissue (Lees, 1991). Na+-K+-ATPase and Ca2+-Mg2+-ATPase are highly vulnerable to free radical inactivation. Thus, we hypothesized that B(a)P exposure is associated with a decrease in Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities and that antioxi-dant treatment is able to prevent this alteration in the rat hippocampus. On the other hand, in neurons, the concen-tration of celluar Ca2+ has an important relationship with neurotransmitter release, neurotransmitter excitability, gene expression and cell death (Neher and Sakaba, 2008;

Gover et al., 2009; Oliveira and Bading, 2011). The con-centration of Ca2+ interacts with the activity of ATPase. Several studies have demonstrated that Ca2+ release can be found in the hippocampus (Manita and Ross, 2009; Salm and Thayer, 2012), so we hypothesized that B(a)P expo-sure is associated with a change of Ca2+ content and that antioxidant could prevent the change.

Some previous studies have demonstrated that Morris water maze (MWM) is an effective method for testing the learning and memory ability of rats. Further-more, male rodents show an advantage in spatial learning (Bucci et al., 1995) and less tolerance to B(a)P in blood elements and organs compared with the females (Knuckles et al., 2001). Hence, male SD rats were cho-sen in this study. B(a)P can induce acute oxidative stress neurotoxicity in the F-344 rat with the dose of 25-200 mg kg−1 body weight and in another study researchers used a repeated design with dosage of 125 mg/kg/day to testify the acute toxicity of B(a)P (Saunders et al., 2006; Torous et al., 2012). In this study, we hypothesized that low-dose B(a)P exposure in sub-chronic toxicity test could also induce spatial learning and memory impairment, that these effects may be associated with changes of oxidative stress, ATPase and Ca2+ content, and that BHA could pre-vent these changes.

MATERIALS AND METHODS

Animals Ninety-six male SD rats (weighing 290-320 g)

aged at seven-weeks were obtained from Chongqing Medical University Lab Animal Center (Chongqing, China) for this study. The rats were housed on a stand-ard 12-hr light/dark cycle (lights on at 8:00 a.m.) at a con-stant temperature of 22°C (± 2°C) and relative humidity of 50% (± 20%) with unlimited (?) access to standard lab-oratory rodent food and tap water. Each rat was marked with a number on their ears using non-toxic ink. All 96 rats were evenly distributed into 16 cages and had a one-week adaptive phase in the same experimental environ-ment. Afterwards, the rats were randomized into four groups. All care and experimental procedures for the rats were conducted in accordance with guidelines published in the Guide for the Care and Use of Laboratory Animals of Chongqing Medical University.

ChemicalsB(a)P (99% purity, Sigma-Aldrich Co., St Louis, MO,

USA) and BHA (99% purity, Sigma-Aldrich Co.) were dissolved in peanut oil (20 mg/mL) which was fresh-ly prepared weekly. Biochemical kits purchased from

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Nanjing Jiancheng Bioengineering Institute (Nanjing, China) were used for the determination of malonalde-hyde (MDA), superoxide dismutase (SOD), Na+-K+-AT-Pase and Ca2+-Mg2+-ATPase. Fura-2/AM calcium fluo-rescent probes were purchased from Sigma-Aldrich Co. Other experiment reagents were obtained from Chong-qing Medicines, Co., Ltd. (Chongqing, China).

Reagent administrationOwing to their poor water solubility, B(a)P and BHA

were prepared by mixing the compound with peanut oil to provide consistent absorption and were subject to son-ication for 30 min at 40°C. The daily reagent dosage was determined individually for each rat based on body weight. B(a)P (2 mg/kg/day) and/or BHA (50 mg/kg/day) were administered to rats daily by intragastric administra-tion for 90 days. Rats were treated between 0800 hr and 1100 hr. The 4 treatment groups were: (A) solvent control group with peanut oil only; (B) BHA group with oral dose of BHA (50 mg/kg/day); (C) B(a)P-exposed group with oral dose of B(a)P (2 mg/kg/day); and (D) B(a)P-BHA-combination group of B(a)P 2 mg/kg/day and BHA 50 mg/kg/day.

Morris Water Maze After 90 consecutive days to B(a)P and BHA, rats

were observed for their performance in MWM. The MWM was a circular pool with 130 cm in diameter, 50 cm in height and was filled with tap water at 22°C ± 1°C to a depth of 30 cm from the brim. Add milk pow-der into the water, and stir it in the pool to make the water opaque. A circular escape platform which was 9 cm in diameter was placed 1.5 cm beneath the surface of the water in the middle of one of the four quadrants of the pool. Through the whole test the position remained the same. A video camera was mounted on the ceiling directly above the pool to record the swim path of each rat using a video tracking system. The room was illuminated by four 30 W spotlights pointed at a white ceiling, and mul-tiple distant cues around the room were kept in the same location throughout the experiments. Rats were placed in a quiet experimental room for a minimum of 30 min prior to testing. Before the MWM test, we needed to test the swimming ability, visual ability and motor activity of each rat. Place the platform in the pool and make sure the top of the platform is above the water surface. Then, put the rat into the water one by one. If the swimming abili-ty and visual ability are normal, rats have no difficulty in swimming to the platform directly. The MWM test con-tained two tasks, which were a hidden platform test and a probe test. Before the hidden platform test, all rats were

first acclimated to MWM in a one-trial habituation proc-ess, which was a 120-sec free swim in the pool without the platform. The hidden platform test began the day after habituation. For each trial, every rat was allowed to escape to the platform for a maximum of 120 sec in the pool. The hidden platform test was carried out for four trials per day in four consecutive days. Latencies to reach the escape platform were recorded. The rats were permitted to have a 30 sec rest on the platform before removal from the pool. If a rat did not reach the platform within 120 sec, it was gently guided to the platform by the experimenter. Rats remained on the platform for 30 sec and were assigned a latency score of 120 sec for that trial. During the trials, the rats were toweled and fan dried and kept in holding cages for at least 5 min.

On day 5 of the MWM test, rats were given a 120-sec probe test of the spatial location without the plat-form. The rats were released from the quadrant opposite from the previous platform quadrant, and the swimming was tracked by an automated tracking system, Morris Maze Experimental Assistant System (Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China), for 120 sec. The number of times for each rat crossed the original platform location and the percentage of time spent in the target quadrant were recorded. Test-ing was performed between 1900 hr and 2300 hr.

Determination of MDA and SOD Following the WMW test, MDA content and SOD

activity were measured to determine the oxidative damage induced by B(a)P and the protective effect of BHA. Eight rats chosen randomly from each group (32 of 96 rats) were anesthetized with pentobarbital-sodium. The brains were removed and bilateral hippocampus tissues were separated and weighed. Next, hippocampus was rinsed in 20 mM Tris-HCl buffer (pH 7.4) and homogenized with ice-cold saline. The mixture was homogenized by glass homogenizers in normal saline. The homogenate was cen-trifuged at 15,000 rpm for 15 min at 4°C and the superna-tants were used to estimate lipid peroxidation by malond-ialdehyde (MDA) content and superoxide dismutase (SOD) activity kit. The concentration of MDA was meas-ured at a wavelength of 532 (Ohkawa et al., 1979). SOD activity in the hippocampus was measured by the meth-od described by Misra and Fridovich at a wavelength of 550 nm (Misra and Fridovich, 1972). The MDA content and the activity of SOD were calculated and normalized to the protein concentration. All analyses were performed as recommended by manufacturers

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Determination of ATPase activityFollowing the WMW test, the activity of Na+-K+-AT-

Pase and Ca2+-Mg2+-ATPase was measured to deter-mine the energy metabolism damage induced by B(a)P and the protective effect of BHA. Eight rats chosen ran-domly from each group (32 of 96 rats) were anesthetised with pentobarbital-sodium. The brains were removed and bilateral hippocampus tissues were obtained and weighed. Next, the hippocampus tissues were separated and homogenized in cold saline to obtain 2% homoge-nates for ATPase activity measurement.

ATPase activity was determined by measuring the rate of formation of phosphoric acid from ATP (Wyse et al., 2000). The process was determined according to the rea-gent kit instruction (Nanjing Jiancheng Bioengineering Institute). The ATPase activity was calculated as: ATPase activity in tissue protein = (A1-A3)/A2*C*D*6/T umol Pi/mg protein, where A1, A2 and A3 are the absorbance of sample, standard and control, respectively. C is the phos-phoric content of standard. D is the dilute times of sample and T is the protein concentration of sample.

Protein content was determined by the Coomassie blue protein-binding method, using bovine serum albumin as standard (Bradford, 1976) .

Determination of Ca2+ concentration Following the WMW test, the Ca2+ content was meas-

ured to detect the damage in the hippocampus induced by B(a)P and the protective effect of BHA. After the behav-ioral experiments, the rest of the rats from each group (32 of 96 rats) were selected for the preparation of synapto-somes and the determination of Ca2+ concentration. The synaptosomes preparation was performed according to the method described by Booth and Clark (1978).

The specific method is as follows. Rats were killed by decapitation and their forebrains were rapidly removed and dropped into ice-cold isolation medium (0.32M-sucrose/ 1 mM-potassium EDTA/10 mM-Tris/HCI, pH 7.4). Next, forebrains were chopped into small pieces and washed repeatedly. The chopped tissue was then homogenized in a Dounce-type glass and the homogenate was dilut-ed to 60 mL with isolation medium and spun at 1,300 g for 3 min in a MSE 18 high-speed centrifuge at 4°C. The supernatant from this spin was centrifuged at 17,000 g for 10 min, producing the crude synaptosomal pellet. After resuspending and diluting, top of isolation medium was layered. The tubes were centrifuged at 99,000 g for 30 min in a 3*23 mL swing-out rotor in a MSE 50 cen-trifuge. Synaptosomes banded at the second interphases. Suck off synaptosome gently and dilute. The final synap-tosomal pellet from rats was taken up in isolation medi-

um in a final volume of 1-2 mL.The synaptosomes was made into turbid liquid with

superfusing with a HEPES-buffered solution, and the pro-tein concentration was adjusted to 2 mg/mL. After 5 min preincubation, 7.5 μL Fura-2/AM (5 μmol/L) and 0.1% bovine serum albumin (BSA) were added in 1.5 mL turbid liquid. After shaking, the mixed liquid was incubated with gas containing 95% oxygen and 5% carbon dioxide for 40 min at 37°C, followed by washing twice with Hanks solution (in mmol/L : NaCl 137.0, CaCl2 1.3, KCl 5.4, MgSO4 0.8, NaHPO4 0.38, KH2PO4 0.44, NaHCO3 4.2, sucrose 5.6, BSA 0.1%; pH 7.4). And the relevant experi-ment reagents were obtained from Chongqing Medicines, Co., Ltd.

The fluorescence value from 1 mL synaptosomes sus-pension was measured by a Hitachi -850 fluorescence spectrophotometer (Hitachi Company, Tokyo, Japan) with excitation wavelength at 335 nm and emission wave-length at 410 nm. Fluorescence intensities were measured continuously and the [Ca2+]i was calculated by the formu-la described as [Ca2+]i = Kd × (F – Fmin )/(Fmax − F), where Kd is the dissociation constant for Fura-2. The F val-ue was the fluorescence intensity measured at with exci-tation wavelengths at 340 nm,and the maximal F value (Fmax) and minimal F value (Fmin) were determined by the addition of 10 μL 10% TritonX-100 (final concentration 0.1%) and 10 μL 500 nmol/L EGTA (final concentration 5 nmol/L), respectively.

Statistical analysisAll data analyses were performed with SPSS v.20.0

(SPSS, Inc., Chicago, IL, USA) and the quantitative data were expressed as mean ± S.D. The data were analyzed using analysis of variance (ANOVA). Data obtained over training days from the hidden platform trial were analyzed by ANOVA for repeated measurement. The remaining data were analyzed by one-way ANOVA. When appropri-ate, post hoc comparisons were assessed using the Least Significant Difference (LSD). A probability of P < 0.05 was considered significant for all analyses.

RESULTS

Morris Water MazeHidden platform MWM

Before the MWM test, all rats in each group could suc-cessfully swim to the platform directly. It was determined that 2 mg/kg B(a)P could not influence the swimming ability, visual ability and motor activity of rats. In the hid-den platform test, the escape latencies for the four groups decreased in a day-dependent manner. There were signif-

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icant main effects of group (F0.05(3,476) =5.855, P < 0.05) and day (F0.05(3,476) = 1790.078, P < 0.05), but no significant group and day interaction (F0.05(3,476) = 1.636; P > 0.05) on the escape latencies. On day one, the escape latencies of rats between groups showed no significant difference (F0.05(3, 92) = 1.062, P = 0.369). But, during days two to four, the post hoc comparisons revealed that the B(a)P-exposed group showed longer latencies to find the platform than the control group (P < 0.05). Also, the B(a)P-exposed group had longer latencies to find the platform than the B(a)P-BHA group (P < 0.05; Fig. 1).

Probe trial The percentage of time in the target quadrant and the

number of times the rats crossed over the location of the hidden platform were measured to assess the spatial mem-ory ability. The one-way ANOVA indicated that there was a significant difference between the four groups in the number of platform crossings and the percentage of time in target quadrant (F0.05 (3,92) = 13.766 and F0.05 (3,92) = 31.996, P < 0.05). The LSD result showed that B(a)P decreased the number of platform crossings and the percentage of time in target quadrant for the significant difference between the quantitative values of control and B(a)P group (P < 0.05, P < 0.05) (Fig. 2A and B). Meanwhile, no dif-ference was found between the control group and the BHA group (P > 0.05) and B(a)P group is significant less than B(a)P-BHA group (P < 0.05) (Fig. 2A and B). Rats of the B(a)P group swam aimlessly in WMW; and rats in con-

trol swam mainly in the target quadrant (Fig. 3A, B, C and D).

The changes of MDA content and SOD activity in hippocampus

The one-way ANOVA indicated significant difference between the groups in MDA level and activity of SOD in rat hippocampus (MDA: F0.05 (3, 28) = 6.335, P < 0.05; SOD: F0.05 (3, 28) =8.213, P < 0.05) (Figs. 4A and B). The LSD result showed that SOD activity decreased signifi-cantly in the B(a)P group compared with the control (P < 0.05), and MDA content increased significantly in B(a)P group compared with the control group (P < 0.05). Mean-

Fig. 1. The escape latencies in hidden platform WMW after exposure to B(a)P (2 mg/kg), BHA (50 mg/kg) and B(a)P-BHA group. Data are shown as mean ± S.D., n = 24, #P < 0.05 versus control (two-way ANOVA fol-lowed by LSD). *P < 0.05 versus B(a)P-exposed group (two-way ANOVA followed by LSD).

Fig. 2. Comparison between effects of exposure to B(a)P (2 mg/kg), BHA (50 mg/kg) and the B(a)P-BHA group in the probe trial. (A) The number of rat platform crossings in the target area. (B) Time in target quadrant of rats (% of 120 sec). Data are shown as mean ± S.D., n = 24, *P < 0.05 versus control (one-way ANOVA followed by LSD). **P < 0.05 versus B(a)P-exposed group (one-way ANOVA followed by LSD).

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while, no differences of the SOD activity and MDA con-tent were found between the control group and the BHA group (P > 0.05, P > 0.05). SOD activity in the B(a)P group was significantly less than that in the B(a)P-BHA group (P < 0.05), and MDA content in the B(a)P group is significant greater than the in the B(a)P-BHA group (P < 0.05) (Fig. 4A and B).

The changes of Na+-K+-ATPase and Ca2+-Mg2+-ATPase activity

The Na+-K+-ATPase and Ca2+-Mg2+-ATPase activi-ty in rat hippocampus between the four groups was sig-nificantly different (Na+-K+-ATPase: F0.05 (3,28) = 43.233,

P < 0.05; Ca2+-Mg2+-ATPase: F0.05 (3,28) = 31.013, P < 0.05). The LSD result showed that the activity of Na+-K+-AT-Pase and Ca2+-Mg2+-ATPase decreased significantly in the B(a)P group compared with the control group (P < 0.05, P < 0.05). Meanwhile, no differences in the activi-ty of Na+-K+-ATPase and Ca2+-Mg2+-ATPase were found between the control group and the BHA group (P > 0.05, P > 0.05) and the activity of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in the B(a)P group was significantly less than that in the B(a)P-BHA group (P < 0.05, P < 0.05) (Fig. 5A and B).

Fig. 3. Representative photographs from the four groups of the rat swimming paths in the probe trial. (A) The control group swim-ming path shows that the rat swims around the target location intensively. (B) The BHA-group (50 mg/kg) swimming path shows that the rat swims around the target location intensively. (C) B(a)P-group (2 mg/kg) swimming path shows the rat swims aimlessly in the pool and spent approximately the same time in the four quadrants. (D) The B(a)P-BHA group swim-ming path shows the rat swims more in the target quadrant compared to (C).

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The changes of Ca2+ content Ca2+ content in rat hippocampus also showed a signifi-

cant difference (F0.05(3,28)= 2.491, P < 0.05). The LSD result shows that Ca2+ content increased in the B(a)P group more than in control group (P < 0.05). Meanwhile, no dif-ference in Ca2+ content was found between the control group and the BHA group (P > 0.05) while the Ca2+ con-tent in the B(a)P group increased significantly compared to the B(a)P-BHA group (P < 0.05) (Fig. 6).

DISCUSSION

The neurotoxic effects of B(a)P have been studied for decades and the mechanism of its toxic effect in the brain is researched in many previous studies (Saunders et al., 2001; Wormley et al., 2004; Niu et al., 2010). One of the mechanisms studied is the relationship between the neurotoxic effect of B(a)P and the hippocampus, which is a particularly vulnerable and sensitive region of the brain. It has a close relationship with the spatial learn-ing, memory and cognitive abilities (Morris et al., 1982;

Fig. 4. Comparison between effects of exposure to B(a)P (2 mg/kg), BHA (50 mg/kg) and the B(a)P-BHA group on the concentra-tion of MDA and the activity of SOD. Data are shown as mean ± S.D., n = 8, *P < 0.05 versus control (one-way ANOVA followed by LSD). **P < 0.05 versus B(a)P-exposed group (one-way ANOVA followed by LSD).

Fig. 5. Comparison between effects of exposure to B(a)P (2 mg/kg), BHA (50 mg/kg) and the B(a)P-BHA group on the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase. Data are shown as mean ± S.D., n = 8, *P < 0.05 versus control (one-way ANOVA followed by LSD). **P < 0.05 versus B(a)P-exposed group (one-way ANOVA followed by LSD).

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Eichenbaum, 1997; Moser et al., 2008). WMW assay demonstrated that B(a)P can impact these abilities. (Morris et al., 1982; Morris, 1984). The studies of expo-sure to B(a)P showed that neuronal damage was found in the hippocampus, spatial learning and memory def-icits associated protein expression signatures (Chen et al., 2012). Behavior was the most significantly affected gene ontology category and learning and memory ranked fourth (Qiu et al., 2011). Most previous studies demon-strated the neurotoxicity of B(a)P based on acute poi-sonous experimental model. In this study, we found that rats in sub-chronic toxicity test exposed to B(a)P (2 mg/kg) had worse learning and memory performance than the solvent control rats. This demonstrated that the neu-rotoxicity of low dosage B(a)P in the sub-chronic toxici-ty experiment model is consistent with the conclusions of previous studies.

Oxidative stress in the hippocampus may have a rela-tionship with the impairment of learning and memo-ry. MDA, an index of oxidative tissue damage, is a by-product of lipid peroxidation induced by free radicals. Accumulation of MDA in intra- and extra-cellular mem-branes will lead to damage to the cells (Cini et al., 1994). SOD, a kind of endogenous antioxidant enzyme, can clear free radicals and have a protective effect in cells (Leadon et al., 1988). In this study, the increase of MDA and the decrease of SOD in the B(a)P-exposed group (2 mg/kg) suggested that B(a)P induces neurobehavioral toxicity by increasing the MDA level and inhibiting SOD activity. It may have a relationship with the inhibition of

antioxidant scavenging system (Saunders et al., 2006). Moreover, energy supply is important to the metab-

olism in the hippocampus. When hippocampus cells are damaged, the type of glycolysis causes the decrease of ATP level, and excess lactate is produced. Follow-ing that, intracellular H+ concentration increases and this may be related to defects in learning, memory and cogni-tion (Siesjö, 1988; Natale et al., 1990). ATPase includes Na+-K+-ATPase and Ca2+-Mg2+-ATPase etc. Thus calci-um overload in cerebral tissues partly results in irrevers-ible brain injury (Palmer et al., 1988; Stys, 1998). In the present study, the sub-chronic exposure to B(a)P caused a decrease in ATPase activity and an increase in Ca2+ con-tent in the hippocampus. These changes are consistent with the previous studies.

Oxygen free radical in brain causes the damage and peroxidation of unsaturated fatty acid on the cell mem-brane, which damages spatial learning and memory abili-ty. Oxidative stress in the brain relates to the spatial learn-ing and memory abilities (Kline et al., 2004). In this study, the concentration of MDA increased significantly after exposure to B(a)P in the hippocampus, which dem-onstrated that lipid peroxidation happened in hippocampal neurons, and neuron damage was produced consequent-ly. When brain damage is caused by oxidative stress, the application of antioxidant can decrease the level of lip-id peroxidation and ameliorate the learning and memory abilities (Row et al., 2003).

BHA is one of the most common stable synthetic phe-nolic antioxidants, and previous studies have demon-strated that BHA has protective capacity in prevent-ing cell death induced by Shikonin (Park et al., 2013) and that BHA plays a role in the oxidative processes in rats (Jeong et al., 2005; Sanchez-Gallego et al., 2011). Wattenberg LW found the protective effect of BHA on chemical carcinogenesis and the change of the chemical carcinogens’ metabolism (Wattenberg, 1986, 1975). In this study, BHA was chosen to prevent the neurotoxicity on oxidative stress caused by B(a)P. In the B(a)P-BHA-com-bination group, the rats had a better performance signifi-cantly in MWM compared to the rats in the B(a)P group. This demonstrates that in behavior tests BHA has protec-tive action against the harmful effect of B(a)P. Further-more, the MDA level of rats in B(a)P-BHA-combination group is lower, and SOD activity is higher obviously than in the B(a)P-group. This is a further sign of the positive effect against B(a)P damage. The activity of ATPase has a significant improvement, which indicates that BHA also plays a positive role in energy metabolism. Regrettably, there is no significant difference of Ca2+ content between the B(a)P group and the B(a)P-BHA-combination group.

Fig. 6. Comparison between effects of exposure to B(a)P (2 mg/kg), BHA (50 mg/kg) and the B(a)P-BHA group on the Ca2+ concentration. Data are shown as mean ± S.D., n = 24, *P < 0.05 versus control (one-way ANO-VA followed by LSD).

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Maybe a larger quantity of BHA can lead to a significant difference.

In conclusion, this study suggests that sub-chronic B(a)P exposure with a lower dose of 2 mg/kg can dam-age learning and memory ability. With the decline of Na+-K+-ATPase and Ca2+-Mg2+-ATPase activity, the accumula-tion of Ca2+ content in endochylema, the decline of SOD activity and the increase of oxygen radicals in hippocam-pus, B(a)P plays a vital role in sub-chronic neurotoxicity process. On the contrary, BHA could prevent these harm-ful effects. This suggests that the neurobehavioral toxici-ty of B(a)P might have a close relationship with oxidative damage, decreasing of ATPase activities and increasing of Ca2+ concentration in the hippocampus. In addition, BHA, as a food additive, widely exists in various types of food and its easy metabolic peculiarity makes it of great value for experimental and clinical applications.

ACKNOWLEDGMENTS

This research was supported by the grant 81372957 of the National Natural Science Foundation of China, PR China.

The authors wish to acknowledge Youbin Qi, Chongying Qiu and Tianyong Wu for their excellent assistance.

Conflict of interest---- The authors declare that there is no conflict of interest.

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