Neurobehavioural effects, redox responses and tissue distribution in rat offspring developmental exposure to BDE-99

Chemosphere 75 (2009) 963–968

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Neurobehavioural effects, redox responses and tissue distribution in rat offspring developmental exposure to BDE-99 Jinping Cheng a,*, Jinmin Gu a, Jing Ma a, Xue Chen a, Muci Zhang b, Wenhua Wang a a b

School of Environmental Science and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China School of Medicine, China Medical University, Shenyang 110001, PR China

a r t i c l e

i n f o

Article history: Received 19 August 2008 Received in revised form 16 December 2008 Accepted 2 January 2009 Available online 8 February 2009 Keywords: BDE-99 Neurobehavioural effects Redox responses Tissue distribution Developmental exposure

a b s t r a c t Polybrominated diphenyl ethers (PBDEs) have recently been shown to be on the increase in the environment and in human milk. The most commonly found PBDE congener in human milk is 2,20 ,4,40 ,5-penta BDE (BDE-99). The aim of the present study was to investigate the neurotoxic effects of BDE-99 (2 mg kg1 d1) administration, from gestational day 6 to postnatal day (PND) 21, on neurobehavioural development and redox responses in offspring. Neurobehavioural development analysis revealed a delayed appearance of cliff drop and negative geotaxis reflexes in the exposed group. Furthermore, developmental exposure to BDE-99 also affected learning and memory functions during adolescence. On PND 37, the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) was reduced, while increases in hydrogen peroxide, lipid peroxidation, nitric oxide and electron spin resonance signal intensities were observed in the hippocampus of BDE-99-treated animals. However, the activity of SOD and GSH-Px in the cerebellum and cerebral cortex was not significantly different between treated and control animals. The present study demonstrated that developmental BDE-99 exposure causes oxidative stress in the hippocampus of offspring by altering the activity of different antioxidant enzymes and producing free radicals. We demonstrated adverse effects of developmental exposure to BDE-99 associated with tissue concentrations very close to the current human body burden of this persistent and bioaccumulative compound. Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.

1. Introduction Polybrominated diphenyl ether (PDBE) mixtures are currently used as flame retardants in various commercial products such as electronic equipment and textiles (De Wit, 2002; Hardy, 2002). Concern about possible adverse effects of PBDEs in humans arose when the levels in Swedish human milk were shown to have increased exponentially over the past few decades, in contrast to pollutants such as polychlorinated biphenyls (Noren and Meironyte, 2000). PBDEs have also been detected in adipose tissue and cord blood (She et al., 2002; Darnerud et al., 2007). The most commonly found PBDE congener in human milk and cord blood is 2,20 ,4,40 ,5penta BDE (BDE-99) (Noren and Meironyte, 2000). Since the human fetus and infant are exposed to BDE-99 by placental transfer and milk, it is important to obtain more information on developmental neurotoxicity. A number of experimental studies have been conducted in mice and rats, in order to evaluate the potential noxious effects of devel-

* Corresponding author. Tel.: +86 21 54742823; fax: +86 21 54740825. E-mail addresses: [email protected], [email protected] (J. Cheng).

opmental exposure to BDE-99 on developing and adult organisms (Eriksson et al., 2001; Branchi et al., 2003). The results indicate that BDE-99 can cause persistent disturbances in spontaneous motor behaviour and dysfunction in learning and memory in adult mice and rats. Nitric oxide (NO), a short-lived free radical generated endogenously by NO synthases, affects a number of physiological functions in the central nervous system, including neurotransmission and learning and memory (Cheng et al., 2005a,b). The hippocampus has long been implicated in memory function in humans and other animals (Venkataraman et al., 2007). However, the effects of BDE-99 on oxidative stress and NO production in brain tissues are not well defined, even though BDE-99, NO and the hippocampus are all related to learning and memory functions. Oxidative stress is one of the most important pathogenic factors related to pollutant toxicity, which can trigger a cascade of events that lead to cell injury and tissue dysfunction in many diseases (Stohs and Bagchi, 1995). The expressions of proto-oncogenes (c-fos/c-jun) are regulated by synaptic activity and play an important role in the neuroplastic mechanisms which are critical to memory consolidation (Shimokawa et al., 2006). The response of proto-oncogenes in the brain to pollutants might be dependent on interaction among NO, neurotransmitter and oxidative stress (Cheng et al., 2005b, 2006a,b). Therefore, the present study ex-

0045-6535/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2009.01.004

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plored the redox responses in offspring developmental exposure to one of the most persistent PBDE congeners, BDE-99. In addition, we assessed the effects of developmental exposure to BDE-99 on juvenile basal motor activity levels and adolescent learning and memory functions, and investigated the tissue distribution of BDE-99 in offspring. Since the brain is not a homogeneous organ and different regions have different functions (Hill and Switzer, 1984), Such as the cerebral cortex plays a central role in many complex brain functions including attention, perceptual awareness, thought, language and consciousness, the cerebellum is involved in the coordination of movement and balance (Venkataraman et al., 2007), we explored the oxidative status of the cerebellum, cerebral cortex and hippocampus. 2. Materials and methods 2.1. Animals and treatment A total of 10 primiparous Sprague–Dawley rats obtained from Shanghai Animal Experimental Center of China Science Institute on gestational day (GD) 1, were housed individually in a room in a 12-h light/12-h dark cycle maintained at 20 ± 3 °C, with free access to food and water. On GD 5, female rats were randomly divided into a control (n = 5) and exposure (n = 5) group. BDE-99 (Promochem, Wesel, Germany) dissolved in corn oil, at a concentration of 2.0 mg mL1, was administered daily to the exposure group by gavage (2.0 mg kg1 d1), from GD 6 to postnatal day (PND) 21, except for PND 0 when dams were left undisturbed. Control animals were dosed with the vehicle only during the same period. Within 24 h of birth, a litter was randomly reduced to six male neonates, which were then maintained by a dam until weaning on PND 21. All pups were euthanized on PND 37. One pup was randomly selected from each treatment group for determination of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and GSH (n = 5). One pup was randomly selected from each treatment group for determination of lipid peroxidation (LPO), H2O2 and NO (n = 5). One pup was randomly selected from each treatment group for electron spin resonance (ESR) measurements (n = 5). The remaining pups in each group were used for chemical analysis (n = 15). Brains were immediately removed and washed repeatedly in ice-cold physiological saline and dissected over ice-cold glass slides to remove the cerebellum, cerebral cortex and hippocampus. The tissues for biochemical analysis and ESR measurements were weighed accurately, and placed in chilled 0.01 M Tris–HCl buffer, pH 7.4. The samples were homogenized and used for determining the biochemical parameters or ESR measurements. Liver and adipose tissue were dissected and washed repeatedly in ice-cold physiological saline. Due to the small size of the pups, the cerebellum, cerebral cortex, hippocampus, live and adipose tissues were pooled on a litter basis and frozen at 80 °C for chemical analysis. The dose of BDE-99 (2 mg kg1) in this study was chosen after considering previous studies on the disruptive effects of PBDE on different behavioural parameters, which indicated a no-observedeffect level between 0.4 and 0.8 mg kg1 (Viberg et al., 2004). The GD 6 to PND 21 period of administration has been chosen because it goes from the formation of the first central nervous system areas (around GD 6) to weaning (PND 21), when the indirect exposure to the compounds through the mother ends (Rice and Barone, 2000). Furthermore, this prolonged developmental exposure is constant and maternally-mediated, like the one occurring in infant humans. Finally, the GD 6-PND 21 administration period has been widely validated as a strategy to assess the neurotoxicity of PCBs and PBDEs in a number of studies (Branchi et al., 2002, 2005; Zhou et al., 2002).

2.2. Neurobehavioural analysis Every 2 d, from PND 3–21, all pups from each litter of each treatment group were used for postnatal assessment of neurobehavioural development (n = 30). Pups were tested according to a slightly modified Fox battery (Branchi et al., 2002); a test procedure intended to provide reliable indicators of neurological and behavioural development. The tests were conducted at approximately the same time of day. The following reflexes were scored: Righting reflex: pup turns over with all 4 feet on the ground when placed on its back; Negative geotaxis: when the pup is placed on a 45° angle slope with his head pointing down the incline, it will turn and crawl up the slope; and Cliff drop test: when the pup is placed on the edge of a cliff with the forepaws and face over the edge, it will turn and crawl away from the cliff drop. Six pups per litter of control and PBDE-treated animals (n = 30) were tested for short-term memory in the Morris water maze (Morris, 1981). The Morris water maze was divided into four equal quadrants. An escape platform was submerged 1cm below the water surface. The water tank was located in a room that contained several visual cues, such as a round mirror and a poster on the wall, and a daylight lamp on the ceiling. All cues were kept constant throughout the experiment. Pups were given four trials per day for three consecutive days from PND 34–36. Each of the four starting points was used once in the experimental day. Once the pup was located on the platform, it was allowed to remain there for 30 s. If the pup did not find the platform within 2 min, it was gently directed to the platform by hand and it was allowed to remain there for 30 s. 2.3. Biochemical analysis The activity of SOD and GSH-Px was estimated by the method of Marklund and Marklund (1974) and 5,50 -dithio-bis-(2-nitrobenzoic acid) (DTNB) photometric method (Rotruck et al., 1973), respectively. GSH and NO contents were measured by the DTNB method (Moron et al., 1979) and enzymatic reduction assay (Kim et al., 2002), respectively. LPO and H2O2 generation were assayed using the method of Devasagayam and Tarachand (1987) and Holland and Storey (1981), respectively. ESR measurements were conducted using a Bruker EMX-spectrometer and a flat cell assembly. Details of the analytical methods for biochemical analysis can be found in our previous studies (Cheng et al., 2005b; Ji et al., 2006). 2.4. Determination of BDE-99 Details of the analytical methods can be found elsewhere (Branchi et al., 2005; Darnerud et al., 2007; Kuriyama et al., 2007). Homogenization, extraction and rough estimation of BDE-99 levels: a sub-sample of tissue was extracted homogenizing it in a 7 + 2 mixture of hexane/isopropanol using an ultra-turrax. The extract was analyzed by capillary gas chromatography with mass spectrometry in single ion detection mode (GC/MS-SIM) leading to approximate concentrations of BDE-99. 2.4.1. Clean-up An internal standard PCB 209 was added to the extract. The mixture was then shaken, centrifuged, decanted and concentrated. For the clean-up, this concentrated extract was poured onto a combination of two SPE-columns. The first SPE-column consisted a combination of silica phase impregnated with sulfuric acid (SiOH–H) and a strongly acidic cation exchanger based on silica with benzenesulphonic acid modification (SA). This combination column is used together with a silica phase (SiOH) column.

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2.4.3. Quality control In each series of determinations (12–15 samples) two controlsamples consisting of bacon fat either spiked with native PBDE or unspiked were analyzed as well. Detection limit: 5 mg kg1 fat. Recovery rate: 100% (determined with spiked bacon fat).

120

Control

*

Escape time (sec)

2.4.2. Instrumental analysis The extract cleaned by the SPE-columns was either diluted or concentrated according to the rough estimation of the concentration of the BDE-99 in the tissue. Instrumental analysis was performed using capillary gas chromatography with an electron capture detector.

*

* *

60

30

0

1-2

3-4

5-6

7-8

9-10

11-12

Trials

2.5. Statistical analysis Statistical analysis was performed using SPSS v. 11.0 (Chicago, IL). Analysis of variance (ANOVA) and Student’s t-test were used to measure differences between groups. The differences were regarded as statistically significant at P < 0.05. 3. Results 3.1. Somatic development BDE-99 did not significantly affect pregnancy duration, proportion of successful deliveries, litter size, and sex ratio. The relative anogenital distance, measured at PND 1, was also not affected by BDE-99 (P > 0.05, data not shown). Pup body weight analysis during the period from PND 1–33 revealed that there were no significant differences between exposed groups and controls (P > 0.05, data not shown). 3.2. Behavioural effects 3.2.1. Effects on neurobehavioural development The righting reflex did not differ between treated and control animals. However, the cliff drop and negative geotaxis reflexes were delayed significantly in exposed offspring relative to controls (P < 0.05, Fig. 1). 3.2.2. Effects on learning and memory In the water maze task (Fig. 2), the time taken to reach the platform for the control and BDE 99-exposed rats shortened as the 100

Percentage of adult-like responses

BDE 99

90

Fig. 2. Latency in reaching the platform in two consecutive trials with Morris’ water maze for each offspring of the BDE 99-treated and control groups. Bars represent mean ± S.E.M., n = 30. *P < 0.01 vs. control group.

number of trials increased (P < 0.01, by ANOVA). There were no significant differences in the escape time between the two groups on the first day (trial 1–4). However, rats that were developmentally exposed to BDE-99 showed a significantly longer latency (P < 0.05) in locating the platform on the second and third days (trial 5–12) of the acquisition period, compared to the control group. 3.3. Biochemical effects 3.3.1. Effects on enzymatic and non-enzymatic antioxidants The specific activity of SOD and GSH-Px was significantly decreased in the hippocampus in the exposed group on PND 37, when compared with the controls (Table 1). However, the activity of SOD and GSH-Px in the cerebellum and cerebral cortex, and the level of GSH in all brain regions did not differ between the treated and control animals (P > 0.05). 3.3.2. Effects on LPO, H2O2 and NO levels Table 1 compares the levels of LPO, H2O2 and NO generation between control and BDE 99-treated groups. Increased levels of LPO, H2O2 and NO generation were observed in different brain regions in BDE 99-treated rats. However, the level of LPO generation in the cerebellum and H2O2 generation in the cerebral cortex did not differ significantly between the exposed groups and controls (P > 0.05).

80 Table 1 Level of SOD, GSH-Px, GSH, LPO, H2O2 and NO in brains of rat offspring on PND 37 following exposure to BDE 99 or vehicle from GD 6 to PND 21.

60

A, Control A, BDE 99 B, Control

40

B, BDE 99 * C, Control C, BDE 99 **

20

0 3

5

7

9

11

13

15

17

19

21

Postnatal day Fig. 1. Neurobehavioural development of pups after developmental exposure to BDE 99 or vehicle. (A) Righting reflex. (B) Cliff drop test. (C) Negative geotaxis. Bars represent mean ± S.E.M., n = 30. *P < 0.05, **P < 0.01 vs. control group.

SOD (U mg1 protein) GSH-Px (U mg1 protein) GSH (nmol mg1 protein) LPO (nmol mg1 protein) H2O2 (lmol mg1 protein) NO (nmol mg1 protein)

Control BDE-99 Control BDE-99 Control BDE-99 Control BDE-99 Control BDE-99 Control BDE-99

Cerebellum

Cerebral cortex

Hippocampus

9.3 ± 1.1 8.9 ± 0.9 140.5 ± 13.3 144.3 ± 16.8 5.8 ± 0.9 4.7 ± 1.0 2.4 ± 0.4 2.6 ± 0.6 2.3 ± 0.3 3.2 ± 0.4* 1.5 ± 0.3 1.7 ± 0.2

7.6 ± 1.1 8.3 ± 0.8 180.1 ± 9.9 173.9 ± 13.6 4.7 ± 0.8 5.2 ± 1.2 1.7 ± 0.4 2.4 ± 0.3* 2.4 ± 0.6 2.6 ± 0.7 1.4 ± 0.4 1.6 ± 0.4

9.8 ± 0.4 7.4 ± 0.6** 165.9 ± 7.8 119.7 ± 5.1** 5.1 ± 1.3 4.9 ± 0.9 0.9 ± 0.1 1.7 ± 0.2** 1.8 ± 0.1 3.1 ± 0.2* 0.9 ± 0.1 2.1 ± 0.2**

Data are presented as mean ± S.E.M., n = 5. * P < 0.05. ** P < 0.01 vs. control group.

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3.3.3. Effects on free radicals ESR spectra of the spin adducts obtained from hippocampal tissues are presented in Fig. 3A. The principal spin adduct had hyperfine splitting constants of aH = 22.7 ± 1.6 G; aN = 15.5 ± 0.5 G. Based upon these constants, it appeared that the principal spin ad duct resulted from the trapping of an alkyl free radical (R ). However, the intensity of the six-line signal was not equivalent, and it was considered to have resulted from coupling with a DMPOOH adduct, with the splitting constants of aH = 15.2 ± 0.4 G; aN = 15.5 ± 0.5 G. Changes in ESR signal intensity are shown in Fig. 3B, in which it was shown that ESR signal intensity in the hippocampus significantly increased compared with that in the controls (P < 0.01). However, the ESR signal intensity in the cerebellum and cerebral cortex did not differ significantly between the exposed groups and controls (P > 0.05). 3.4. Tissue levels of BDE-99 BDE-99 persisted in the brain, liver and adipose tissue of offspring until puberty. BDE-99 in the brain, adipose and liver of exposed rats was significantly higher than that in the control group (P < 0.01, Table 2). When comparing levels of BDE-99 in these tissues, the highest concentrations were found in adipose tissue, followed by liver, hippocampus, cerebellum and cerebral cortex. 4. Discussion The present study showed that developmental BDE-99 exposure affected several end points measured from birth to 37 d of age. Analysis of developmental landmarks and postnatal reflex responses revealed that developmental BDE-99 exposure did not grossly impair somatic development of the offspring. Nevertheless,

A Control Hippocampus

BDE 99 Hippocampus

ESR signal intensities (% of control)

B 180 160 140 120 100 80 60 40 20 0

100%

there was a significantly delayed appearance of the cliff drop and negative geotaxis reflexes in the BDE-99 group. Furthermore, developmental exposure to BDE-99 also affected learning and memory functions in adolescent rat offspring. Other groups have reported neurodevelopmental disturbances in rodents exposed to PBDEs, and this system seems to be the most sensitive to PBDE-induced toxicity. Supporting our data, Branchi et al. (2002, 2003) have found that developmental exposure to BDE-99 produces several behavioural alterations, for example, a significantly delayed appearance of the climbing response. Kuriyama et al. (2005) have found that the cliff drop aversion reflex is significantly delayed following developmental exposure to BDE-99. Eriksson et al. (2001) have found that neonatal exposure to BDE-99 or BDE-47 disrupts neurobehavioural responses and causes hyperactivity in mice that appears to be permanent and worsens with age. Viberg et al. (2004) have found that neonatal exposure to PBDEs disrupts normal behaviour, impairs learning and memory. The crucial role of the hippocampus during brain development is well known. Disturbance of the hippocampus can cause serious impairment in neurological development and learning and memory functions (Porterfield, 1994). The cliff drop, negative geotaxis reflex responses and Morris water maze tests showed offspring developmentally exposed to BDE-99 performed significantly worse than control animals (Figs. 1 and 2). This is similar to the impairment in spatial learning tasks that have been seen in aged rodents in the Morris water maze (Gage et al., 1984). Spatial learning is one form of memory in which humans also show significant impairment with ageing (Viberg et al., 2003). This indicates that developmental BDE-99 exposure can accelerate ageing process. The responses of spatial learning in this present study may reflect dysfunction in the hippocampus tissues. Further biochemical studies showed that developmentally exposed to BDE-99 gave rise to different changes in antioxidants in different brain regions. The levels of SOD, GSH-Px GSH, NO and ESR signal intensity in the cerebellum and cerebral cortex were not significantly different between treated and control animals. However, Developmental exposure to BDE-99 caused significant changes in SOD, GSH-Px, LPO, H2O2, NO and ESR signal intensity in the hippocampus (Table 1), which indicates that the hippocampus in adolescent animals is a target for developmental neurotoxicological effects of BDE-99. However, it is different from MeHg toxicity. There is clear evidence that cerebellum is as a possible target for MeHg toxicity. Autopsy studies in MeHg-exposed humans found the cerebellum to have the highest levels of total mercury in the brain, and cerebellar damage is often reported following MeHg exposure in humans and rodents (Lapham et al., 1995; Pedersen et al., 1999). Antioxidant enzymes are an essential part of the cellular defence against reactive oxygen species (ROS). Decreased activity of antioxidant enzymes (SOD, GSH-Px) and increased level of LPO and H2O2 generation (Table 1) in the hippocampus of BDE-99-treated animals indicate an increase in oxidative stress in the hippocampus (Venkataraman et al., 2007). In support of our findings, previous studies have reported that PBDE causes oxidative stress, DNA damage and apoptosis, by

Table 2 BDE-99 levels in the brain, liver and adipose tissue of adolescent rat offspring at 37 d of age (ng g1 wet weight, mean ± S.E.M., n = 5).

Cerebellum

Cerebral cortex

Hippocampus

Fig. 3. Effect on free radicals. (A) ESR spectra obtained from the hippocampus tissue within 5 min of the reaction. # shows the position of the DMPO-OH signal; * shows the position of the DMPO-R signal. (B) ESR signals intensity in the brain of rat offspring on PND 37 following exposure to BDE 99 or vehicle from GD 6 to PND 21. Bars represent mean ± S.E.M., n = 5. **P < 0.01 vs. control group.

Cerebral cortex Cerebellum Hippocampus Adipose Liver **

Control

BDE 99

0.4 ± 0.1 0.3 ± 0.1 0.5 ± 0.2 2.3 ± 0.9 0.6 ± 0.2

230.4 ± 43.8** 242.7 ± 58.1** 261.6 ± 65.8** 2576.3 ± 351.6** 634.7 ± 125.5**

Significant difference between exposed groups and control P < 0.01.

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10000 2576.3 1380

ng/g

1000

398 74.4

100 10 1 Adipose

BDE-99

BDE-99

Σ PBDE

Σ PBDE

tissues

mean

max

mean

max

Our study

Human adipose tissues level

Fig. 4. Concentration of BDE-99 in adipose tissues in the present study compared to accumulation of BDE-99 and RPBDE in human adipose tissue samples from New York City. Human mean and maximum values were taken from Johnson-Restrepo et al. (2005).

decreasing the enzyme activity of GSH-Px and SOD and/or increasing ROS production in primary rat hippocampal neurons (He et al., 2008). Mandavilli and Rao (1996) and Venkataraman et al. (2007) have shown that the hippocampus is more susceptible to oxidative damage when compared to the cerebellum and cerebral cortex. Oxidative stress is the cytotoxic consequence of oxygen radical and oxidant formation and their reaction with cellular constituents. The hippocampus, as with many other tissues, has a range of antioxidant defences, which help to maintain redox status (Venkataraman et al., 2007). NO, a highly reactive free radical, acts as both a physiological messenger, like a neurotransmitter, and a neurotoxic agent in the central nervous system (Ji et al., 2006). In the present study, ESR findings and NO bioassay results showed that NO level and free radical signal intensity in the hippocampus of exposed group were markedly increased. Excess NO can react  rapidly with superoxide radical (O2 ) to form a potent and powerful  long-lived oxidant, peroxynitrite (ONOO ). This can interact with nucleic acids, proteins and lipids and contribute substantially to the cellular redox state (Radi et al., 2001). SOD protects against  oxygen free radicals by catalyzing the removal of O2 , which damages the membrane and biological structures (Venkataraman et al., 2004). The decrease in SOD activity in the hippocampus (Table 1) following exposure to BDE-99 was associated with increased levels of free radicals (Fig. 3). GSH-Px metabolizes peroxides such as H2O2 and protects cell membranes from LPO. GSH-Px is also involved in scavenging of H2O2 (Ji et al., 2006). The observed decline in the activity of GSH-Px in the hippocampus of BDE-99-exposed animals may be ascribed to an increase in the level of peroxides (Table 1). GSH plays a major role in controlling cellular redox state, and this is a primary mechanism for H2O2 and peroxide removal in the brain (Venkataraman et al., 2007). The increased H2O2 in brain regions may also be due to the lack of changes in GSH level (Table 1). Increased production of malondialdehyde is regarded as an indicator of LPO. LPO is one of the main manifestations of oxidative damage and has been found to play an important role in toxicity in many vertebrates (Venkataraman et al., 2007). The increased LPO in the hippocampus may also have been caused by decreased SOD activity (Table 1). The decrease in SOD activity in the hippocampus might have resulted in greater accumulation of free radicals. It could have been an auto-destructive mechanism in the hippocampus. This might have upset the pro-oxidant/antioxidant balance within the hippocampus, which could be one of the main reasons for the increase in oxidative stress in the hippocampus of the BDE-99-exposed group. The reduction of the capacity of the major antioxidants and the excess availability of free radicals in

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the hippocampus indicated aggravation of oxidative status following developmental exposure to BDE-99. The redox responses of BDE-99 can be ascribed to pharmacokinetic mechanisms, as suggested by the results of analytical studies. When comparing levels of BDE-99 in different brain regions, the highest concentrations were found in hippocampus, followed cerebellum and cerebral cortex (Table 2). The results more confirm that hippocampus is as a possible target for BDE-99 toxicity. Significant amounts of BDE-99 were found in adolescent offspring tissue, which clearly indicated that internal exposure to BDE-99 continued after puberty (Table 2). This is in agreement with the slow elimination rate and bioaccumulation previously reported for BDE-99 (Hakk and Letcher, 2003). The terminal elimination halflife for PBDE-99 in humans has been calculated as 2.9 years (Geyer et al., 2004). In adult rat adipose tissue, the half-life has been calculated as 41.6 d in females (Geyer et al., 2004). In our study, although we could not calculate the half-life, the high tissue concentration of BDE-99 found on PND 37 corroborated the data from Geyer et al. (2004) and Kuriyama et al. (2007), which suggest a long half-life for BDE-99 in rats. We compared the adipose tissue concentration of BDE-99 in rat offspring in the present study with the levels of BDE-99 and RPBDE, respectively, in adipose tissue of humans (Fig. 4). The values of BDE-99 and RPBDE in human adipose tissue were taken from the study of Johnson-Restrepo et al. (2005). The adipose tissue concentrations achieved in the present study were approximately 34.6 (BDE-99) and 6.5 (RPBDE) times, respectively, higher than the average concentration of BDE-99 and RPBDE found in human adipose tissue. The adipose tissue concentration of BDE-99 in rat offspring was only 1.9 times higher than the maximum reported level of BDE-99 in non-occupationally exposed humans (Johnson-Restrepo et al., 2005). In conclusion, the results of this study suggest that developmental BDE-99 exposure causes serious impairment in neurological development and learning and memory functions in developing rats. The hippocampus is more susceptible to oxidative damage when compared to the cerebellum and cerebral cortex. The present study demonstrated that developmental BDE-99 exposure causes oxidative stress in the hippocampus of rat offspring by altering the activity of different antioxidant enzymes and producing free radicals. Many factors may be involved in the mechanisms of BDE-99 neurotoxicity. We have demonstrated adverse effects following developmental exposure to BDE-99, associated with tissue concentrations very close to the current human body burden of this persistent bioaccumulative compound. Acknowledgements The authors acknowledge the financial support of Chinese National Natural Science Foundation (20607014). References Branchi, I., Alleva, E., Costa, L.G., 2002. Effects of perinatal exposure to a polybrominated diphenyl ether (PBDE 99) on mouse neurobehavioural development. Neurotoxicology 23, 375–384. Branchi, I., Capone, F., Alleva, E., Costa, L.G., 2003. Polybrominated diphenyl ethers: neurobehavioral effects following developmental exposure. Neurotoxicology 24, 449–462. Branchi, I., Capone, F., Vitalone, A., Madia, F., Santucci, D., Alleva, E., Costa, L.G., 2005. Early developmental exposure to BDE 99 or Aroclor 1254 affects neurobehavioural profile: interference from the administration route. Neurotoxicology 26, 183–192. Cheng, J., Wang, W., Jia, J., Zheng, M., Shi, W., Lin, X., 2006a. Expression of c-fos in rat brain as a prelude marker of central nervous system injury in response to methylmercury-stimulation. Biomed. Environ. Sci. 19, 67–72. Cheng, J., Wang, W., Qu, L., Jia, J., Zheng, M., Ji, X., Yuan, T., 2005a. Rice from mercury contaminated areas in Guizhou province induces c-jun expression in rat brain. Biomed. Environ. Sci. 18, 96–102. Cheng, J., Yuan, T., Wang, W., Jia, J., Lin, X., Qu, L., Ding, Z., 2006b. Mercury pollution in two typical areas in Guizhou province, China and its neurotoxic effects in the

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