Jun expression and tissue distribution in rat offspring prenatally co-exposed to MeHg and PFOA: PFOA impairs Hg retention

Jun expression and tissue distribution in rat offspring prenatally co-exposed to MeHg and PFOA: PFOA impairs Hg retention

Chemosphere 91 (2013) 758–764 Contents lists available at SciVerse ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere ...

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Chemosphere 91 (2013) 758–764

Contents lists available at SciVerse ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Neurobehavioral effects, c-Fos/Jun expression and tissue distribution in rat offspring prenatally co-exposed to MeHg and PFOA: PFOA impairs Hg retention Jinping Cheng a,⇑, Masatake Fujimura b, Wenchang Zhao c, Wenhua Wang a a b c

School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, China Department of Basic Medical Sciences, National Institute for Minamata Disease, Minamata, Kumamoto 867-0008, Japan School of Environmental and Safety Engineering, Changzhou University, Changzhou, Jiangsu 213164, China

h i g h l i g h t s " Co-exposure to MeHg and PFOA significantly induced cliff avoidance. " PFOA was antagonistic to MeHg toxicity in the locomotor activity test. " Co-exposure to MeHg and PFOA decreased all tissue Hg concentrations in pups. " PFOA impaired Hg retention in different tissues.

a r t i c l e

i n f o

Article history: Received 4 August 2012 Received in revised form 2 February 2013 Accepted 9 February 2013 Available online 12 March 2013 Keywords: Methylmercury Perfluorooctanoic acid c-Fos/Jun expression Locomotor activity test

a b s t r a c t Exposure to methylmercury (MeHg) and perfluorooctanoic acid (PFOA) can occur simultaneously as both contaminants are found in the same food sources, especially fish, seafood, marine mammals and milk. The aim of this study was to assess the effects of exposure to MeHg (10 lg mL 1 in drinking water) and PFOA (10 lg mL 1 in drinking water) from gestational day 1 to postnatal day (PND) 21, alone and in combination, on neurobehavioral development and the expression of c-Fos/Jun in different brain regions in the offspring. Our findings showed that exposure to MeHg alone, and exposure to MeHg combined with PFOA significantly induced cliff avoidance reflexes and negative geotaxis reflexes. And these effects appeared to be greater following exposure to MeHg alone. MeHg and/or PFOA exposure did not significantly impair motor coordination functions, or cause significant changes in c-Fos expression in the hippocampus and cerebellum, and spatial learning tests were similar to those in the controls, thus it was impossible to determine whether combined exposure to MeHg and PFOA had any additional effects on both hippocampus and cerebellum regions. However, a significant increase in the frequency of line crossing was observed in rats treated with MeHg or PFOA alone, and there were no significant differences between the MeHg + PFOA-treated group and the controls, suggesting that PFOA was antagonistic to MeHg toxicity in the locomotor activity test. Co-exposure to MeHg and PFOA decreased all tissue Hg concentrations in pups compared to the group exposed to MeHg only, suggesting that PFOA impaired Hg retention in different tissues. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Methylmercury (MeHg) is one of the most ubiquitous environmental contaminants. Due to the recycling of Hg through the environment, ingestion of small quantities of Hg due to consumption of contaminated fresh water fish and seafood is likely (Knobeloch et al., 2007; Ulrich et al., 2007; Shao et al., 2011). Perfluorinated ⇑ Corresponding author. Address: School of Environmental Science and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China. Tel.: +86 21 54742823/13916873206. E-mail addresses: [email protected], [email protected] (J. Cheng). 0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.02.016

compounds (PFCs) are a class of emerging persistent contaminants. These chemicals are fully fluorinated man-made chemicals with unique properties which make them useful in a wide array of industrial and household applications such as refrigerants, surfactants and polymers, and as components of pharmaceuticals, fire retardants, lubricants, adhesives, cosmetics, paper coatings, and insecticides (Houde et al., 2006; Lau et al., 2007; Liao et al., 2009; Gellrich et al., 2012). The wide use of PFCs in a variety of products has also resulted in them being ubiquitous in the environment, where they occur globally in humans and wildlife (Lindh et al., 2012). Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) have mostly received worldwide attention because they are ubiquitous

J. Cheng et al. / Chemosphere 91 (2013) 758–764

and found in wildlife and humans, as well as being found/used in the production of degradation products in everyday commercial applications (Olsen et al., 2005; Fromme et al., 2009). In humans, PFOS and PFOA have been detected in serum samples from the general population in the USA and Canada (Hansen et al., 2001; Ostertag et al., 2009), in human milk and blood samples in China (So et al., 2006; Yeung et al., 2006) (e.g., PFOS, 45–360 ng L 1, PFOA, 47–210 ng L 1 in milk; PFOS, 52.7 ng mL 1, PFOA, 1.59 ng mL 1 in blood samples) and in human serum in Japan (Harada et al., 2007; Liu et al., 2007). Several food web studies have shown that some PFCs have the potential to bioaccumulate in lower trophiclevel organisms, and through tropic transfer and biomagnification, can accumulate in upper trophic level organisms (Van de Vijver et al., 2003). While exposure pathways of PFOA, PFOS and related PFCs in humans have not yet been fully elucidated, the consumption of fish (Falandysz et al., 2006) and farm animals (Guruge et al., 2005) has been suggested as a major contributor of PFCs in exposed human populations (Yoo et al., 2009). Therefore, exposure to PFOA and MeHg can occur simultaneously as both contaminants are found in the same food sources, especially fish, seafood, marine mammals and milk. A number of experimental studies have been conducted in mice and rats in order to evaluate the potential noxious effects of exposure to MeHg in developing and adult organisms (Yasutake and Hirayama, 1988; Sakamoto et al., 2002). There is increasing evidence to show that PFOA/PFOS exposure can induce various toxic effects in animals such as hepatotoxicity, developmental, reproductive and systemic toxicity, interference in thyroid hormone level, mitochondrial bioenergetics and cell–cell communication, neuroendocrine dysfunction and carcinogenicity (Hu et al., 2002; Lau et al., 2004; Liao et al., 2009). However, there is still a paucity of information on the potential additive, synergistic or antagonistic interactions between MeHg and PFOA. Therefore, the specific aims of this study were: (1) to investigate whether co-exposure to MeHg and PFOA can interact and enhance developmental neurotoxic effects; (2) to determine whether MeHg and PFOA alter c-Fos/c-Jun expression in the cerebellum, cerebral cortex and hippocampus of adolescent pups; (3) to investigate if co-exposure to MeHg and PFOA can affect levels of Hg in different tissues. Immediate early genes (IEGs) including c-fos and c-jun in neurons are easily induced by a variety of extracellular stimuli (Usuki et al., 2000, 2008; Dong et al., 2005). In the normal condition, c-fos and c-jun genes have low expression in the nerve cell and take part in cell growth, cell polarization and message transfer. External stimulation such as pollutants, cold or lack of blood in the brain can change the expression of IEGs (Fujimura et al., 2000a,b). It has also been reported that IEGs take part in the MeHg-induced neurotoxicity process. Expression of the IEG product has been used as a marker of the induced neuronal activation. The laboratory results obtained in this study will enhance the understanding of neurotoxic effects induced by MeHg and/or PFOA and provide the basic information of exposure and the risk assessment for human beings (Cheng et al., 2005a,b, 2006a, 2009a; Usuki et al., 2008).

2. Materials and methods 2.1. Animals and treatments All experimental procedures involving animals were performed in compliance with the National Institute of Minamata Disease (NIMD) on the care and use of laboratory animals. Wistar rats (nine females and nine males, 10 weeks old) were supplied by CLEA Japan. Amphimixis occurred after 3 d of acclimatization. Females were inspected daily for the presence of a vaginal plug (gestational day, GD 0). On GD 1, the males were removed and the nine females

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were randomly divided in four treatment groups (Control, MeHg, PFOA, MeHg + PFOA) as indicated below: MeHg treatment: MeHg dissolved at a concentration of 10 lg mL 1 (10 ppm) was administered daily to rats (n = 4) in their drinking water from GD 1 to postnatal day (PND) 21. Two of these animals were also exposed to PFOA as indicated below. PFOA treatment: PFOA (Promochem, Japan, Lot WKG6398) dissolved at a concentration of 10 lg mL 1 (10 ppm) was administered daily to rats (n = 5) in their drinking water from GD 1 to PND 21. Control animals (n = 2) were administered with vehicle during the same period. The doses of PFOA and MeHg (10 ppm) were chosen in accordance with previously published reports, Which were the lowestobserved-adverse-effect-level (Sakamoto et al., 2002; Rosen et al., 2007). The GD 1 to PND 21 period of administration was chosen because it ranged from the formation of the first central nervous system areas (around GD 6) to weaning (PND 21), when indirect exposure to these compounds via the mother ends (Rice and Barone, 2000; Cheng et al., 2009b). Within 24 h of birth, a litter was randomly reduced to 11–13 neonates, which were then maintained by a dam until weaning on PND 21. On PND 40, two male and two female pups randomly selected from each litter in each group were euthanized. One male and one female pup were randomly selected from these animals for the determination of c-Fos and c-Jun expression. The remaining male and female pup in each group was used for Hg analysis. The pups for Hg analysis were perfused via the ascending aorta with phosphate buffer after the blood sample was collected. All brains were immediately removed and dissected over ice-cold glass slides to remove the cerebellum, cerebral cortex and hippocampus. The liver and kidney samples were also collected and washed repeatedly in ice-cold physiological saline for Hg analysis. The tissues for Hg analysis were frozen at 20 °C, and the tissues for gene measurement were frozen by immersion in liquid-nitrogen cooled and stored at 80 °C until assayed. 2.2. Assessment of pre-weaning neurobehavioral development Every 2 d, from PND 3 to 21, six pups were randomly selected from each litter in each group and were used for postnatal assessment of neurobehavioral development. The following reflexes were scored (Branchi et al., 2002): Righting reflex: the pup was placed on its back on a flat surface and the time taken to turn over with all four paws was recorded, using a cut-off time of 2 s. Cliff avoidance: the pup was positioned on the edge of a bench, with its forepaws and nose just over the edge. The time taken to withdraw its head and both forefeet was recorded, using a cut-off time of 2 s. Negative geotaxis: the pup was placed on a 45° angle slope with its head downwards, and the percent success rate and the time necessary to turn around 180° were recorded, using a cut-off time of 5 s. 2.3. Assessment of post-weaning neurobehavioral development 2.3.1. Motor coordination The rotarod test was performed to evaluate coordination and balance, with three trials per day for two consecutive days from PND 34-35. The apparatus (Natsume, Tokyo, Japan) consisted of a bar, 8 cm in diameter and 10 cm long, which rotated at 15 rpm. The duration time, that is, the time from when the pup was mounted on the rod until it fell off, was recorded in seconds. Individual performance was assessed using a cut off of 60 s (Sakamoto et al., 2002). All pups were tested. 2.3.2. Locomotor activity Locomotor activity was assessed using an open field on PND 36, for which the methodological details are given elsewhere (Yoshida

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et al., 2004). Briefly, each pup was moved from its home cage to the center square (5 cm  5 cm) of the open field (15 cm  15 cm), and covered with an opaque Plexiglass box (15 cm  15 cm  15 cm) for 5 min. The behavior of the pup was video-recorded for the following 5 min. The video image was analyzed by Image OF, software for image analysis (O’hara Co. & LTO, Tokyo, Japan). The number of line crossings and central square entries were scored. All pups were tested. 2.3.3. Spatial learning To assess spatial learning and memory, all pups were tested in the Y-maze on PND 39. Each pup was placed at the end of one arm (arm A) facing the center of the maze, and allowed to move freely within the maze for a period of 3 min. The total number of arms entered and the order of arm entries were recorded. The total number of arms entered provides an indication of locomotor activity, and the order of arm entries provides a measure of spontaneous alternation behavior and thus working memory (Podhorna and Brown, 2002). 2.4. Western blot analysis Western blot analyses were performed as described in our former reports (Fujimura et al., 2009, 2012). Brain tissues were prepared by sonication for 5 s in cell lysis buffer (T-Per Mammalian Protein Extraction Reagent, Pierce Biotechnology, Rockford, USA) containing protease and protein phosphatase inhibitors (Sigma–Aldrich, St Louis, USA). Samples were centrifuged (14 000g for 1 h) and the supernatants were collected. Protein content was measured using the DC Protein Assay Kit II (Bio-Rad Laboratories, Hercules, USA). The cell lysates (20 g of protein) were separated by SDS–PAGE (10%) (Tefco, Tokyo, Japan) and transferred to nitrocellulose membranes (GE Healthcare, Buckinghamshire, England). Changes in c-Fos and c-Jun were investigated using the following monoclonal antibodies which were diluted using SDS–PAGE: cFos (GeneTex Inc., San Antonio, USA) and c-Jun (Cell Signaling Technology, Beverly, USA). Proteins were detected using a chemiluminescence system with ECL (GE Healthcare). Densitometric quantification of immunoblots was carried out using Quantity One (version 4.6.3) software (Bio-Rad Laboratories).

Table 1 Reproductive parameters following perinatal exposure to MeHg, PFOA, MeHg + PFOA or vehicle. Parameters

Control

MeHg

PFOA

MeHg + PFOA

Pregnancy rate Gestation length (d)

2/2 21

2/3 21

3/3 20.7 ± 0.7

2/3 20.7 ± 0.5

Litter size Mean ± S.E.M. Range; number

13.5 ± 0.5 13–14

14 ± 2 12– 16 14:14

14.3 ± 0.9 13–16

17 ± 2 15–19

22:21

15:19

The ratio of male/female pups

16:21

No significant differences between exposed groups and controls, p > 0.05.

genital distance, measured at PND 1, was not affected by MeHg or/and PFOA (p > 0.05). 3.2. Behavioral effects 3.2.1. Effects on reflex maturation The righting reflex did not differ among the groups (data not shown). However, the cliff avoidance and negative geotaxis reflexes were significantly delayed in both MeHg and MeHg + PFOA exposed offspring relative to controls (Fig. 1, p < 0.05; v2-test). 3.2.2. Effects on motor coordination, locomotor activity and learning and memory In the motor coordination test (Table 2), male or/and female offspring exposed to MeHg, PFOA, or their combination had a lower success rate compared to the controls. In the locomotor activity test, a significant increase in the frequency of line crossing was observed in the MeHg or PFOA treated rats (Table 2, p < 0.05, by ttest), however, there were no significant differences between the MeHg + PFOA-treated group and the controls. There were no differences among the groups for either gender in central square entries

(A) Cliff avoidance 100 80

2.5. Hg analysis Total mercury concentrations were determined according to the oxygen combustion-gold amalgamation method using a mercury analyzer MA 2000 (Nippon Instruments, Tokyo, Japan). Details of the analytical methods for Hg can be found in our previous studies (Cheng et al., 2009a; Fujimura et al., 2012).

60 40 20 0

The v test and repeated measure ANOVA were used for statistical analysis of the data from the reflex development and rotarod tests. Student’s t-test was used to analyze the data on locomotor activity, learning and memory, gene expression and Hg concentrations.

1

3

5

7

9

11

13

15

17

17

19

19

21

Postnatal day

2.6. Statistical analysis 2

Control MeHg * PFOA MeHg+PFOA *

(B) Negative geotaxis 100 Control

80

MeHg *

60

PFOA * MeHg+PFOA *

40 20

3. Results 3.1. Effects on somatic development Dams in all exposure groups had gestational lengths, litter sizes, percent male pups, and percent live births similar to the controls (Table 1). No deaths were recorded. In addition, the relative ano-

0

1

3

5

7

9

11

13

15

21

Postnatal day Fig. 1. Neurobehavioural development of pups perinatally exposed to MeHg, PFOA, MeHg + PFOA or vehicle. Bars represent mean ± S.E.M. (A) Cliff avoidance test. (B) Negative geotaxis reflexes. Data represent the mean ± SEM. n = 12 pups/group.  p < 0 .05 vs. control group by v2 test.

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J. Cheng et al. / Chemosphere 91 (2013) 758–764 Table 2 Effects on motor coordination, locomotor activity and learning and memory of offspring developmental exposure to MeHg, PFOA, or their association. Behavioral effects

Male

Female

Male + female

Control MeHg PFOA MeHg + PFOA Control MeHg PFOA MeHg + PFOA Control MeHg PFOA MeHg + PFOA

Motor coordinationa

Locomotor activity (frequency)b

Learning and memory (Y-Maze)c

Success rate (%)

Line crossing

Central square entries

Total entries/number

Correct arm entries (%)

92.9 ± 7.2 58.0 ± 16.1 79.4 ± 2.4 59.4 ± 8.4 87.5 ± 12.5 55.0 ± 5.0 69.5 ± 2.8 83.3 ± 0 90.2 ± 6.1 57.0 ± 7.0a2 74.5 ± 2.8a1 70.8 ± 8.0

59.7 ± 3.9 83.0 ± 5.7a1,b2 70.2 ± 3.3a2,b2 53.7 ± 0.4 72.6 ± 5.7 78.9 ± 4.3b2 70.8 ± 5.1a2 57.6 ± 5.7 64.9 ± 3.5 80.8 ± 3.5a1,b2 70.5 ± 2.9b2,c1 55.6 ± 3.8

3.2 ± 0.4 3 ± 0.6 2.5 ± 0.4 3.6 ± 0.7 3.5 ± 0.6 3.9 ± 0.4 2.6 ± 0.4 3 ± 0.5 3.3 ± 0.3 3.5 ± 0.4 2.5 ± 0.3 3.3 ± 0.4

20.6 ± 1.4 23.3 ± 1.1 22.3 ± 1.1 19.8 ± 1.5 21 ± 1.6 20.6 ± 1.6 22.2 ± 1.2 17.8 ± 1.2 20.8 ± 1.1 21.8 ± 1 22.2 ± 0.8 18.8 ± 1

70.7 ± 3.7 75.3 ± 1.7 71.9 ± 3.2 62.3 ± 5.2 66.9 ± 4.7 68.5 ± 3 74.5 ± 2.2 76.5 ± 3 69.2 ± 2.9 71.6 ± 1.9 73.1 ± 2 69.4 ± 3.3

a

Percentages of offspring reaching the criterion of 60 s on rotarod rotating at 15 rpm with six trials repeated. The number of line crossing and central square entries of offspring in the open field for 5 min. Total entries and percentage correct arm entries made in the Y-maze. Data represent the mean ± S.E.M. n = 11–19 male or/and 10–16 female pups/group. a1 p < 0.05. a2 p < 0.01 vs. Control. b2 p < 0.01 vs. MeHg + PFOA. c1 p < 0.05 vs. MeHg.

b

c

Table 3 Relative intensity of c-Fos and c-Jun expression in brain regions of offspring developmental exposure to MeHg, PFOA, or their association from GD 1 to PND 21. Protein expression

Male

Female

Male + female

c-Fos

Control MeHg PFOA MeHg + PFOA Control MeHg PFOA MeHg + PFOA Control MeHg PFOA MeHg + PFOA

c-Jun

Cerebellum

Cerebral cortex

Hippocampus

Cerebellum

Cerebral cortex

Hippocampus

100 ± 13.5 116.4 ± 4.2 94.6 ± 7.9 108.2 ± 0 100 ± 3.1 83.4 ± 8.2 93.1 ± 2.4 83.0 ± 8.2 100 ± 5.9 99.3 ± 8.3 93.8 ± 3.8 95.1 ± 6.2

100 ± 8.1 141.1 ± 6 109 ± 5.3 112.1 ± 1.2 100 ± 13.2 127.9 ± 8.8 107.8 ± 12 106.5 ± 22.9 100 ± 6.3 134.5 ± 5.7a2 108.4 ± 6 109.3 ± 9.5

100 ± 7.9 108.6 ± 6.2 100.6 ± 3.5 39.9 ± 28.6 100 ± 8.4 105 ± 12.7 77.1 ± 3 84.7 ± 26.6 100 ± 8.6 107 ± 10 90.3 ± 9.8 59.5 ± 18.3

100 ± 11.2 109 ± 17.4 183.6 ± 8.6a2 169.0 ± 42.4 100 ± 9.3 86.6 ± 0.3 109.4 ± 4.3 88.1 ± 1.4 100 ± 20.2 94.1 ± 13.3 134.1 ± 6.2 115 ± 11.6

100 ± 7.8 119.8 ± 7.2 112.6 ± 2.4 127.3 ± 0.3a1 100 ± 6 125.2 ± 10.6 124.1 ± 13.2 107.6 ± 7 100 ± 4.1 122.5 ± 5.9a2 118.4 ± 6.9 117.3 ± 5.5a1

100 ± 5.7 103.1 ± 3.5 103.2 ± 1.5 78.2 ± 6.1 100 ± 9.7 95.3 ± 13 129.8 ± 4.2a1 133.5 ± 7.7 100 ± 25 97.5 ± 23.1 122.1 ± 28 117.5 ± 42

n = 2–3 male or/and 2–3 female pups/group. p < 0.05. p < 0.01 vs. Control by t-test.

a1 a2

A

B

C

Fig. 2. Representative blots of western blots. (A) Cerebellum; (B) cerebral cortex; (C) hippocampus.

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Table 4 T-Hg levels in the tissues of rat offspring (ng/g wet weight, mean ± S.E.M).

male

Female

Male + female

Control MeHg MeHg + PFOA Control MeHg MeHg + PFOA Control MeHg MeHg + PFOA

Blood

Liver

Kidney

Cerebellum

Cerebral cortex

Hippocampus

0.47 ± 0.06 1.43 ± 0.14a1 1.04 ± 0.19 0.61 ± 0.29 1.56 ± 0.06a1,b1 0.92 ± 0.13 0.54 ± 0.13 1.49 ± 0.07a2,b2 0.98 ± 0.10a1

0.51 ± 0.18 1.09 ± 0.05 0.77 ± 0.17 0.45 ± 0.12 1.14 ± 0.01a1 0.78 ± 0.19 0.48 ± 0.09 1.11 ± 0.02a2,b1 0.78 ± 0.10

0.62 ± 0.22 2.97 ± 0.00a2 2.36 ± 0.52 0.60 ± 0.17 3.32 ± 0.03a2 2.86 ± 0.20a1 0.61 ± 0.11 3.14 ± 0.10a2 2.61 ± 0.27a2

0.34 ± 0.06 0.75 ± 0.01a1 0.57 ± 0.16 0.40 ± 0.12 0.72 ± 0.03a1,b2 0.39 ± 0.00 0.37 ± 0.05 0.74 ± 0.01a2,b1 0.48 ± 0.10

0.41 ± 0.02 0.64 ± 0.03a1 0.58 ± 0.16 0.39 ± 0.04 0.77 ± 0.05a1 0.56 ± 0.13 0.40 ± 0.02 0.71 ± 0.05a2 0.57 ± 0.09

0.33 ± 0.01 0.80 ± 0.00a2 0.62 ± 0.17 0.39 ± 0.05 0.75 ± 0.04a1 0.52 ± 0.09 0.36 ± 0.03 0.77 ± 0.02a2 0.57 ± 0.08

n = 2–3 male or/and 2–3 female pups/group. p < 0.05. a2 p < 0.01 vs. control. b1 p < 0.05 vs. MeHg + PFOA by t-test. b2 p < 0.01 vs. MeHg + PFOA by t-test. a1

in the locomotor activity test and in learning and memory in the Ymaze (p > 0.05, by t-test, Table 2). 3.3. Effects on expression of c-Fos and c-Jun There were no significant differences among the groups for either gender in c-Fos expression in different brain regions in rat offspring exposed to MeHg, PFOA, or their combination (p > 0.05 by t-test, Table 3 and Fig. 2). The common finding in offspring exposed to PFOA was induced c-Jun expression in all brain regions when compared with the controls. However, the changes of c-Jun expression were found in different brain regions when compared with the corresponding controls. 3.4. Tissue levels of Hg T-Hg levels in different tissues in the offspring are shown in Table 4. T-Hg levels in the blood, liver, kidney, cerebellum, cerebral cortex and hippocampus of MeHg-treated offspring significantly increased compared with those in the controls (p < 0.05, by t-test). However, Hg levels in these tissues did not differ significantly between the MeHg + PFOA-exposed groups and controls (p > 0.05). The level of Hg in all tissues in pups given MeHg + PFOA was lower than that in pups given MeHg alone. 4. Discussion The present study showed that prenatal exposure to MeHg or PFOA alone significantly induced the delayed appearance of negative geotaxis reflexes (Fig. 1), impaired motor coordination functions, significantly increased the frequency of line crossing (Table 2), and alterations in c-Fos/Jun expression in the cerebellum, cerebral cortex and hippocampus (Table 3). However, co-exposure to MeHg and PFOA had a minor effect on locomotor activity, but no significant exacerbated effect was seen in co-exposed animals when compared with the controls, and there were significant differences between the MeHg + PFOA-treated group and the MeHg or PFOA exposed groups (p < 0.01). Of interest is that some epidemiological studies have suggested that boys may be more susceptible than girls to the neuropathological effects of MeHg exposure (Goulet et al., 2003), however, in the present study, the neurobehavioral effects, c-Fos/Jun expression and tissue distribution were not significantly different between male and female offspring. The delayed appearance of the cliff avoidance and negative geotaxis reflexes are in agreement with previous reports of developmental exposure to MeHg (Fujimura et al., 2012). Because exposure to MeHg, PFOA, or their combination dramatically induced the delayed appearance of the negative geotaxis reflexes, it was impossible to determine whether combined exposure to MeHg

and PFOA had any additional effect on reflex maturation. Both exposure to MeHg alone and MeHg combined with PFOA significantly induced the cliff avoidance and negative geotaxis reflexes. However, these effects appeared to be greater with MeHg alone (Fig. 1). The cerebellum has an important role in mediating certain aspects of motor function, including balance and coordination. It is known that rats with maldevelopment of the cerebellum induced by neonatal exposure to alcohol show a deficit in motor coordination (Goodlett et al., 1991). In the present study, MeHg and/or PFOA exposure did not significantly impair motor coordination functions (p > 0.05, Table 2), however, a non-significant trend was observed for both PFOA and MeHg + PFOA exposed rats to have slightly higher success rates than MeHg-treated rats. Thus, it is unlikely that combined exposure would exacerbate the effect of MeHg on the cerebellum. The Y-maze test may reflect the ability of recent memory, and multiple brain regions including the hippocampus and amygdala may be associated with this memory function (Podhorna and Brown, 2002). c-Fos is also related to learning and memory functions (Cheng et al., 2005a, 2009a; Usuki et al., 2000, 2008). In the present study, MeHg and/or PFOA did not cause significant changes in c-Fos expression in the hippocampus and cerebellum, and the spatial learning tests in exposed groups were similar to those in the controls. Similarly, it was impossible to determine whether combined exposure to MeHg and PFOA had any additional effect on both hippocampus and cerebellum regions. Locomotor activity in the open field is difficult to interpret because its precise neurobehavioral significance is not well understood (Goulet et al., 2003). Lesion studies suggest that the nucleus accumbens mediates both locomotor activity and exploration, whereas limbic structures (amygdala, hippocampal formation, and prelimbic frontal cortex) modulate the behavioral response to novelty rather than locomotor activity itself (Burns et al., 1996). In the present study, a significant increase in the frequency of line crossing was observed in the MeHg or PFOA alone treated rats (Table 2, p < 0.05, by t-test), however, there were no significant differences between the MeHg + PFOA-treated group and controls, suggesting that PFOA is antagonistic to MeHg toxicity in locomotor activity tests. These results seemed to reflect an interaction due to MeHg and PFOA co-exposure and impaired developmental neurotoxic effects on locomotor activity. In the present study, there were no significant differences among the groups for either gender regarding c-Fos expression in different brain regions. It has been reported that the expression of c-fos and c-jun induced by pollutants was a dynamic process. Previous studies reported that induction of c-Fos in the periaqueductal gray, neocortex and thalamic nuclei by MK-801 was doseand time-dependent, occurring within 2 h and dissipating by 24 h

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after administration (0.5–8.0 mg/kg, i.p.; Dragunow and Faull, 1990; Hattori et al., 2004). Matsuoka et al. (1997) found that the expression of c-fos in cells treated with 20 lM HgCl2 began to increase after 30 min, peaked at 1 h and then returned to the control level at 8 h, the accumulation of c-Fos protein was observed at 1 h and peaked at 2–3 h, but the c-Fos protein was still detected after 8 h exposure to HgCl2. Our previous studies also showed that induction of c-Jun in the different brain regions by MeHg was doseand time-dependent, occurring within 20 min, peaked at 60 min and restoring after 1440 min (Cheng et al., 2006a, 2009a). Based upon these findings, we believe that the expression of c-Fos in different brain regions returned to normal levels after weaning for 19 d on PND 40. In the present study, PFOA significantly induced c-Jun expression in male cerebellum and female hippocampus. The increase in c-Jun expression in the hippocampus has a strong link with impaired learning and memory induced by Hg or PCBs in rats (Cheng et al., 2005b, 2006b, 2009b). However, there were no differences among the groups in the Y-maze test in this study; more corroborating brain regions including the hippocampus may be associated with Y-maze memory function. Although we cannot say whether there is a causal relationship between increased or decreased c-Jun expression and neurobehavioral development, these results show that further investigations are necessary. Altogether, these results indicate that non-additive interactions occurred between MeHg and PFOA regarding the effects on c-Fos and c-Jun expression in the cerebellum, cerebral cortex and hippocampus, PFOA was significantly antagonistic to MeHg toxicity in the locomotor activity test. The combined effect of MeHg and PFOA can be explained by pharmacokinetic mechanisms, as suggested by the results of the analytical studies. Indeed, blood, liver, kidney, cerebellum, cerebral cortex and hippocampus tissue Hg levels were lower in the co-exposed pups than in those treated with MeHg alone (Table 4). PFOA impaired Hg retention in different tissues. Acknowledgments The authors thank the reviewers of this article for their thoughtful suggestions and valuable insights. The work was financially supported by a General Program from the National Natural Science Foundation of China (No. 21177087) and a Grant from the Major State Basic Research Development Program of China (973 Program) (No. 2013CB430005). 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. Burns, L.H., Annett, L., Kelley, A.E., Everitt, B.J., Robbins, T.W., 1996. Effects of lesions to amygdala, ventral subiculum, medial prefrontal cortex, and nucleus accumbens on the reaction to novelty: implication for limbic–striatal interactions. Behav. Neurosci. 110, 60–73. 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., Yang, L., Hu, W., Zheng, M., Wang, W., Liu, X., Qu, L., 2005b. Neurobiological disruptions induced in brains of the rats fed with mercury contaminated rice collected from experimental fields in Guizhou Province, China. Chin. Sci. Bull. 50, 2441–2447. 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., 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 brains of rats fed with local polluted rice. Environ. Geochem. Health 28, 499– 507. Cheng, J., Yang, Y., Ma, J., Wang, W., Liu, X., Sakamoto, M., Qu, Y., Shi, Wei, 2009a. Assessing noxious effects of dietary exposure to methylmercury, PCBs and Se coexisting in environmentally contaminated rice in male mice. Environ. Int. 35, 619–625.

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