Maternal exposure to environmental bisphenol A impairs the neurons in hippocampus across generations

Toxicology 432 (2020) 152393

Contents lists available at ScienceDirect

Toxicology journal homepage: www.elsevier.com/locate/toxicol

Maternal exposure to environmental bisphenol A impairs the neurons in hippocampus across generations

T

Haibin Zhanga,1, Zhouyu Wanga,1, Lingxue Menga, Hongxuan Kuanga, Jian Liua, Xuejing Lva, Qihua Panga, Ruifang Fana,b,* a

Guangdong Provincial Engineering Technology Research Center for Drug and Food Biological Resources Processing and Comprehensive Utilization, School of Life Sciences, South China Normal University, Guangzhou, 510631, China b Guangdong Provincial Key Laboratory of Chemical Pollution and Environmental Safety, South China Normal University, Guangzhou 510006, China

ARTICLE INFO

ABSTRACT

Keywords: Bisphenol A Learning and memory DNA damage Neuron quantities Neurotransmitters Offspring

Humans from fetal to adult stages are chronically and passively exposed to bisphenol A (BPA, an endocrine disruptor) due to its ubiquitous existence in daily life. To investigate the long-term neurotoxicity of maternal exposure to BPA for offspring, mice were used as the animal model. In this study, pregnant mice (F0) were orally dosed with BPA (i.e. mice from low-, medium- and high-exposed groups were treated with 0.5, 50, 5000 μg/ kg·bw of BPA per day) until weaning. Then, the first generation (F1) mice were used to generate the F2 ones. The offspring of mice not exposed to BPA served as the control groups. The Y-maze test, comet assay, hematoxylineosin (HE) staining method, Golgi-Cox assay and liquid chromatography-tandem mass spectrometry (LC/MS/ MS) were conducted to study any alterations to learning and memory abilities, the morphological variations in hippocampal neurons and transmitter levels of F1 and F2 mice induced by BPA exposure. Results showed that even a low-dose of maternal BPA exposure could sex-dependently and significantly impair the learning and memory ability of F1 male mice, but not of generation F2. Furthermore, decreased neuron quantities and spine densities in hippocampi were observed in both F1 and F2 generations after maternal BPA exposure. However, DNA damage of brain cells were only limited to F1 offspring, in which DNA damage was only observed in the low-exposed male mice and medium-exposed female mice. Additionally, maternal BPA exposure leads to variations in hippocampal neurotransmitter levels, indicated by the decreased ratio of Glu/GABA in F1 offspring. In conclusion, maternal exposure to an environmental dose of BPA resulted in lasting adverse effects on neurological development for offspring mice.

1. Introduction Bisphenol A (BPA) is widely used in the production of plastics, canned food lining, dental sealants, thermal papers and many other daily necessities (Fan et al., 2015; Geens et al., 2011; Vandenberg et al., 2007). BPA can leach into foods and the environment during the manufacturing process or daily use (Munguia-Lopez and Soto-Valdez, 2001). Due to its ubiquity in our environment, people are inevitably exposed to BPA during their daily lives (Richter et al., 2007; Vom Saal et al., 2007). Biomonitoring studies have detected BPA in many other biological specimens, including milk, maternal amniotic fluid, placental tissue and follicle fluid (Ikezuki et al., 2002; Vandenberg et al., 2010; Vom Saal

et al., 2007). Urinary BPA concentrations ranged from 0.1 to 2.4 μg/ kg·bw in the general population (Ikezuki et al., 2002; Vandenberg et al., 2010; Vom Saal et al., 2007). These studies suggest that humans are exposed to environmental levels of BPA from fetal to adult stages. Studies also show that BPA can penetrate the blood-brain barrier (Sun et al., 2002), influencing developing nervous systems and behaviors (Anderson et al., 2013; Luo et al., 2013; Xu et al., 2011a). “Low-dose” BPA used in this study is equivalent to human exposure BPA levels in daily life, and much lower than the reference dosage (RfD, 50 μg/kg·bw) given by U.S. Environmental Protection Agency (U.S. EPA, 2012) or the acceptable estimated daily intake (t-TDI, 4 μg/kg·bw) recently set by European Food Safety Authority (European Food Safety Authority (EFSA, 2015). They are thought as “safety dosages”. However, many

Corresponding author at: Guangdong Provincial Engineering Technology Research Center for Drug and Food Biological Resources Processing and Comprehensive Utilization, School of Life Sciences, South China Normal University, Guangzhou, 510631, China. E-mail address: [email protected] (R. Fan). 1 These authors contributed equally to this study. ⁎

https://doi.org/10.1016/j.tox.2020.152393 Received 30 August 2019; Received in revised form 30 January 2020; Accepted 31 January 2020 Available online 03 February 2020 0300-483X/ © 2020 Elsevier B.V. All rights reserved.

Toxicology 432 (2020) 152393

H. Zhang, et al.

scientists question these benchmarks as many epidemiological BPA researches showed that BPA exposure under these benchmarks were associated with negative health outcomes (Li et al., 2018; Stein et al., 2015). Endogenous hormones play pivotal roles in sculpting the networks which underlie behavior patterns during brain development (Beatty, 1979; Bonthuis et al., 2010; McCarthy, 2008). Epidemiological and animal studies indicate that BPA exposure during early developmental periods impacts the nervous system and behavior patterns (Anderson et al., 2013; Cox et al., 2010; Gore et al., 2018; Li et al., 2018; Stein et al., 2015; Tewar et al., 2016). In response, a number of countries have issued regulations to ban or limit the use of BPA in consumer products, particularly in baby bottles (European Food Safety Authority (EFSA, 2015.; Government of Canada, 2010; Michałowicz et al., 2014; Food and Drug Administration (FDA, 2012). Besides in baby bottles or toddlers’ toys, BPA has been detected in maternal amniotic fluid, placental tissue, follicular fluid, and milks (Meeker et al., 2009; Sasaki et al., 2005; Vandenberg et al., 2010). Its chronic neurotoxic effects on the following generations via maternal route are not well understood. Generally, BPA’s genotoxicity is controversial. As an environmental endocrine disruptor, BPA binds to the estrogen receptors leading to a series of subsequent physiological actions (Cravedi et al., 2007) The reproductive toxicities of BPA exposure showed that BPA exposure could exhibit transgenerational reproductive effects. For instance, prenatal exposure to BPA could reduce sperm count and motility in males. These phenotypes could even be transmitted to the F3 generation (Bernal and Jirtle, 2010). Some experiments also found that BPA exposure could lead to DNA damage in the peripheral blood and insulinoma cells of rats (Ulutaş et al., 2011; Xin et al., 2014), suggesting the genotoxicity of BPA. Alternatively, in vitro studies indicated that BPA was not genotoxic for human liver, kidney and intestine cells (Audebert et al., 2011). It is unclear whether maternal exposure to lowdosage BPA can exhibit multi- or transgenerational effects on central nervous system and learning and memory abilities. As neurotransmitter and hippocampus play important roles in the behavior and the ability of learning and memory (Myhrer, 2003; Xin et al., 2018; Xu et al., 2011a), we provide a hypothesis whether the neurotoxicity induced by maternal BPA exposure is genotoxic through impairing the balance of neurotransmitter levels and influencing neuron morphology. In this study, the ability of learning and memory, the hippocampal morphology and neurotransmitter levels in the F1 and F2 offspring were studied to investigate neurotoxicity and its genotoxicty in offspring resulting from maternal BPA exposure.

Glu-D5, acetylcholine (ACh), ACh-D9, γ-aminobutyric acid (GABA) and GABA-D6 were purchased from Toronto Research Chemicals (Ontario, Canada). Tea oil, also named camellia Oil (Q/BBAH0025S) was purchased from Yihai (Guangzhou) Grains&Oils Industries Co., LTD (Guangzhou, China). All chemicals were equal to or above analytical grade.

2. Materials and methods

A Y-maze test was used to test the learning and memory abilities of offspring according to the method used in our previous study (Zhou et al., 2017). Each mouse performed 20 trials at 25−35 s every 24 h. For each trial, a discrimination error was recorded as “×” whenever a mouse entered the dark arms where it would receive brief electric foot shocks (30 V AC, 0.45 mA; started at 5 s after lamp-light) until it chose

2.2. The animals studied and the treatment procedures KM mice (breeders, male mice aged 8 weeks with 40 ± 2 g weight and female mice aged 6 weeks with 30 ± 2 g weight, SCXK (Yue) 20110029) were obtained from the Experimental Animals Center of Sun YatSan University. The rearing conditions were controlled at 25 ± 1℃ and with 12-h light/dark cycles. Foods and water were supplied ad libitum. The housed cages and water bottles used in this study were all BPA-free. All mice had three days of acclimatization prior to mating treatment. After the acclimatization period, the mice were randomly divided into four groups. For mating, 2 female and 1 male were housed together in a cage. Pregnant Female mice (designated F0 generation) were randomly divided into four groups (N = 13 mice in each group) and chronically received treatments of different BPA concentrations (i.e. 0, 0.5, 50, and 5000 μg/kg·bw/day for the control, low-, medium- and high-exposed groups, respectively; dissolved in tea oil) administered orally from gestational day 1 (GD 1) until postnatal day 21 (PND 21). The low-exposed mice were treated with 0.5 μg/kg·bw/day BPA according to the average urinary levels measured in the general population (Battal et al., 2014; Chouhan et al., 2014). The medium-exposed mice were treated with 50 μg/kg·bw/day BPA according to the RfD given by EPA (U.S. EPA, 2012), and the high-exposed group was treated with 100 times of the RfD BPA (i.e. 5000 μg/kg·bw/day). On PND 28, mice of the first generation (F1) were randomly divided into subgroups. One subgroup (N = 10–12 male or female mice) first performed the Ymaze tests and then were sacrificed. Their brains and hippocampi were taken out and separated for the Comet assay, HE staining, Golgi-Cox staining and LC/MS/MS analysis. On PND 42, the other part of male and female mice in F1 who were generated from the same F0 exposedgroup was mated to create generation F2. The F2 offspring were used for the same experiments on PND 28. Those F2 mice were not orally administered BPA. The detailed data about litter size and sex ratio of F1 and F2 mice in the Supplemental Table 1. The route diagram of this study was shown in the Supplemental Fig. 1. 2.3. Y-maze test

2.1. Chemical reagents BPA was purchased from Sigma-Aldrich (St. Louis, MO, USA, purity: 98 %). 5-hydroxtryptamine (5-HT), 5-HT-D4, L-glutamic acid (L-Glu), L-

Table 1 Comprehensive comparison of Y maze for F1 and F2 mice after different maternal BPA treatments. Group (μg/kg·bw)

F1

F2

0 0.5 50 5000 0 0.5 50 5000

Trails to qualify the standard 1

Male

Female

p

110.9 ± 4.1 163.3 ± 9.5** 148.3 ± 7.6** 150.0 ± 11.1** 124.0 ± 6.5 134.0 ± 10.8 124.0 ± 9.8 128.0 ± 8.5

133.3 ± 6.2 125.0 ± 7.4 148.3 ± 9.7 121.7 ± 6.7 120.0 ± 6.7 112.0 ± 4.4 130.0 ± 13.1 132.0 ± 10.0

0.012 0.001 1.000 0.015 0.757 0.092 0.642 0.757

The training date of correct reactions above 90 % (day)

The training date of qualifying the leaning standard (day)

Male

Female

Male

Female

4th 7th 6th 7th 4th 6th 4th 5th

4th 5th 6th 4th 4th 4th 4th 4th

7th 11th 9th 11th 8th 9th 9th 9th

8th 9th 11th 8th 7th 7th 10th 9th

There were 12 males and 12 females for each group in F1, and 10 males and 10 females in F2. * or ** indicates that there was a significant difference between the control and exposed groups (p < 0.05 or p < 0.01). p1 when compared between male and female mice. 2

Toxicology 432 (2020) 152393

H. Zhang, et al.

Fig. 1. Effects of maternal BPA exposure on DNA damage of brain cells in offspring. (A) Typical comet assay images of brain cells in F1 offspring in groups. (B) Tail DNA%, tail length and tail moment of brain cells in F1 mice after different BPA treatments (*p < 0.05, **p < 0.01, compared with the control group).

procedures were referred to the previous studies (Iso et al., 2006; Zhou et al., 2017). Briefly, the tail DNA%, tail length, tail moment, the comet percentage and arbitrary units were recorded and measured using CASP (the comet analysis software of Beijing Biolaunching Technologies Co) to determine the full extent of DNA damage.

(recorded as “√”) to enter the bright arm. After training, data were collected, including the trials required to qualify the learning benchmark, the ratio of correct reactions (the percentage of correct trials in 20 trials per day) and the ratio of qualified mice (the percentage of mice qualifying for the learning criterion in a group). A 90 % correct ratio or more for a consecutive 3 days was set as the learning benchmark. In generation F1, 12 female and male mice from each group were tested. In generation F2, 10 female and male mice from each group of F2 were tested. Abnormal mice were abandoned, including mice with a ratio of correct reactions above 80 % on the first training day and those who met the benchmark during the 4 training days or could not meet the benchmark within 10 days.

2.5. HE method Eighteen mice (male and female = 9:9) from each group of F1, and 12 mice (male and female = 6:6) from each group of generation F2 were sacrificed. The HE method was performed according to a procedure identical with previous studies in order to quantify the number of neurons in the hippocampus (Li et al., 2015; Zhou et al., 2017). In brief, after deep anesthesia by intraperitoneal injection with 2 % sodium pentobarbital (0.07 mL/20 g), heart of a mouse was perfused through with normal saline and 4 % paraformaldehyde. Then, entire brain was collected as the sample. After dehydration in gradually evaluated concentrations of ethanol-water solvents, the samples were fixed by

2.4. Comet assay Comet assay was used to detect DNA damage in whole brain cells induced by BPA exposure. 16 mice (male and female = 8:8) from each group of F1 and F2 generations were scarified. The detailed operation 3

Toxicology 432 (2020) 152393

H. Zhang, et al.

paraformaldehyde (pH = 7.4) and embedded in paraffin. Finally, the brain samples were sectioned coronally and serially at 4 μm by automatic rotary microtome. 20 serial slices contained classic hippocampus were randomly obtained from each brain sample. Regions of CA1, CA3 (cornu ammonis 3) and DG (dentate gyrus) were imaged at 200-fold magnification with a Leica wide field microscope (DM6, Leica, Germany). The framed box (186 × 124 mm2) was added to the same place on specific regions in the hippocampal slice pictures. To reduce objective discrimination, the numbers of neurons were counted twice by a blinded method and the average number was used as the final result.

average correct ratios above 90 % on the 7th, 6th and 7th training day, one or two days later than the control F1 male mice. The exposed groups of F1 male mice needed an additional 2 or 4 days to qualify the learning standard compared with the control mice. In generation F2, though no statistical differences in training trials were observed between the male exposed and control groups, mice from the low-exposed group needed more trails to qualify the standard (Table 1). Interestingly, sex difference was observed between F1 control and BPA- exposed mice. In the control group, F1 female mice needed more trials to qualify the standard compared with male mice (p < 0.05). However, after different BPA treatments, particularly after the low- and high-BPA treatments, the situations were reversed, i.e. F1 male mice needed more trials to qualify for the learning standard compared with F1 female mice (p < 0.01).

2.6. Golgi-Cox staining method A modified Golgi-Cox staining method was used for the analysis of hippocampal dendrites and spines (Liu et al., 2016; Zaqout and Kaindl, 2016). In brief, the brain was impregnated in Golgi-Cox solution for two days at 37 ℃, and then transferred into a tissue-protectant solution prior to paraffin embedding. Brains were coronally sectioned at 150 μm by automatic rotary microtome (RM2255, Leica, Germany) and the sections were collected on 5 % gelatin-coated slides. Then the slides were stained in ammonia, immersed with sodium thiosulfate, dehydrated, and cleared until mounting. Then, the hippocampal neurons in CA1 region (N = 6 mice per group and n = 5–10 neurons selected randomly per mice) were imaged at 200-fold magnification, and the spines were imaged at 400-fold magnification with a Leica wide field microscope (Shown in Fig. 2A, B) (Kimura et al., 2016). Sholl analysis was used to analyze the dendritic branching pattern complexity via counting the intersection numbers between dendrites by using an overlaid concentric sphere at 10-μm intervals. Neuron J Plugin was used to quantify the total lengths and branch numbers. The spines along the dendritic segments (N = 6 mice per group, n = 10–20 segments per mice) were counted, and spine density values of CA1 neurons were expressed as the number of spines per 10-μm dendrite.

3.2. BPA effects on DNA damage of brain cells The outcomes of BPA exposure-related DNA damage in brain cells for F1 and F2 mice are presented in Table 2. Comet assay analysis indicated that the comet percentage of brain cells in F1 offspring increased sharply from 24.3 % in the control group to 32.6 % in the lowexposed group, regardless of the sex. Moreover, the arbitrary units (DNA damage scores) of the brain cells of the low-exposed group were much higher than those of the control group of F1 offspring (Table 2). Sex differences of F1 mice existed in the tail DNA%, tail length and tail moment of brain cells after BPA exposure. Significant discrepancies in tail DNA%, tail length and tail moment of brain cells were observed between the F1 control and low-exposed male mice. However, the increased tail DNA%, tail length and tail moment of brain cells in the lowexposed female mice were not statistically different with the control group of generation F1 mice. The medium-BPA treatment significantly increased the tail DNA%, tail length and tail moment of brain cells in F1 female mice (p < 0.01) (Fig. 1). However, no significant difference in DNA damage was observed between the BPA treated and the control groups in the generation F2 mice (Table 2).

2.7. Determination of Glu, GABA, ACh, 5-HT and DA concentrations in hippocampus by LC/MS/MS

3.3. BPA effects on neuron numbers in hippocampus

Neurotransmitter concentrations were determined using a LC/MS/ MS method (Kuang et al., 2019). 100 μL internal isotope standards were added into the samples before protein precipitation with 1 % formic acid in 1 mL ACN (V:V). Supernatants were then taken out for drying under gentle nitrogen. Finally, the residue was reconstituted for LC/ MS/MS (Thermo Scientific, Rodano, Italy) analysis. The limits of qualification for Glu, GABA, ACh and 5-HT were 1.0, 0.50 L, 0.15 and 0.60 μg/L, respectively. The accuracy ranged from 92.9 %–119.2 % with inter-day and intra-day precision of 0.39 %–13.55 %. There were 9–12 mice in each group for determination of neurotransmitters.

Results of hematoxylin-eosin (HE) staining and the representative hippocampal neuron images in CA1, CA3 and DG regions of offspring are presented in Fig. 2A, B. As shown in Fig. 2, the hippocampal neurons of the control group were arranged compactly, while the neurons tend to be dispersed in the BPA-treatment groups. Further analysis indicated that neuron numbers in the hippocampus decreased after BPA exposure, particularly after the medium- and high-BPA exposure treatments. In DG regions of the medium- and high-exposed mice of F1 and F2, neuron numbers were significantly decreased (Fig. 2C, D). In CA3 region, significantly decreased neuron numbers were observed in the medium- and high-exposed male mice of F1 and F2 generations, as well as in high-exposed female mice of F1 and F2 generations (Fig. 2C, D). In CA1 region, though maternal BPA exposure did not significantly impact the neuron numbers of F1 male mice, the F2 male mice exhibited significantly decreased trends. We also observed significantly decreased neuron numbers in the medium- and high-exposed female mice of F1.

2.8. Statistical analysis All statistical analysis was performed by SPSS (SPSS, version 19.0, Chicago, IL). All data were collected and analyzed by a blinded observer, and expressed as mean ± SEM. Two-way ANOVA with dose and sex as the main factors was used for data analysis. Difference between groups was then assessed with Fisher’s exact post hoc tests, which adjust significance levels to take multiple comparisons into account. The significant levels were set at p < 0.05 and p < 0.01.

3.4. BPA effects on dendrites and spines in hippocampus

3. Results

Dendrites and spines in the hippocampus are critical carriers for learning and memory function. Thus, the neural morphology in CA1 was investigated. As shown in Fig. 3A, BPA treatments generally decreased the complexity of CA1 neurons in F1 male offspring. Analysis of the dendrites showed that BPA exposure could significantly decrease the numbers of CA1 dendritic intersections. Compared with those from the control groups, F1 male mice from the low- and high-exposed groups had a significantly decreased number of intersections of CA1

3.1. BPA effects on spatial learning and memory ability The effects of BPA exposure on Y-maze performance are presented in Table 1. Except for female mice, the trials required by F1 exposed male mice to qualify the standard were significantly increased (p < 0.01). Three BPA-exposed groups of F1 male mice reached the 4

Toxicology 432 (2020) 152393

H. Zhang, et al.

Fig. 2. Representative images of CA1, CA3 and DG regions in the hippocampus of F1 (A) and F2 (B) male and female mice. Neuron numbers of CA1, CA3 and DG regions in the hippocampus of F1 (C) and F2 (D) mice. Bar: 100 μm (*p < 0.05, **p < 0.01, compared with the control group). There were 9 male mice and 9 female mice in each group for F1. There were 6 male mice and 6 female mice in each group for F2.

neurons at each concentric circle ranging from 60 to 130 and 30−160 μm away from the cell body. We also observed significantly decreased CA1 dendritic intersections in the F1 medium-exposed male mice (Fig. 3B). Moreover, the total length of CA1 basal and apical dendrites were significantly decreased after BPA exposure (p < 0.01) (Fig. 3C). Investigation of the CA1 dendritic spines showed the spine densities of male offspring decreased remarkably after maternal BPA exposure (Fig. 4). In low-exposed F1 mice, significantly decreased CA1 spine densities were observed (p < 0.01), and the effect exhibited in F1 male mice could even be observed in F2 male, but not in female mice (Fig. 4F). Medium- and high- BPA exposures led to distinctive differences between sexes (p < 0.05). Male but not female mice had a significant decrease in CA1 dendritic spines. This phenomenon was also

observed in mice of generation F2 (Fig. 4F). 3.5. BPA effects on neurotransmitters levels in hippocampus Concentrations of Glu, GABA, ACh, 5-HT and DA, as well as the ratio of Glu/GABA in the hippocampus are shown in Table 3. Generally, BPA treatments increased the concentration of the neurotransmitters and decreased the Glu/GABA ratio in the hippocampi of F1 mice. However, there were sex differences in the neurotransmitter variations between male and female mice. The Glu levels in F1 female mice from BPAexposed groups were significantly higher than those from the control group (p < 0.01). However, the significantly increased Glu levels were not found in F1 exposed male mice (Table 3). Moreover, even the low 5

Toxicology 432 (2020) 152393

H. Zhang, et al.

learning and memory (Bannerman et al., 2014; Moser et al., 2014), and the decreased hippocampal neuron quantities and dendritic spine densities are negatively impact learning and memory processes (Kimura et al., 2016; Liu et al., 2016). As these impaired indexes observed in the F2 mice were not accompanied with significant altered behavior in the Y-maze (Table 1), it conversely indicated that the character change induced by BPA exposure was not stable and might not persist across generations. Furthermore, the Y-maze test is a whole animal behavior experiment, and the performances of mice are related to a multi-step dynamic process in the nervous system, including proliferation, differentiation, migration, expansion of axons and dendrites, synapse formation, myelination, and programmed cell death (Rice and Barone, 2000). Wolstenholme’s exposure route and doses of BPA were very similar to ours, and their results showed that maternal BPA exposure (5000 μg/kg/day) could cause deficits in social recognition and decreased the expressions of post-synaptic densities (PSD) genes, including PSD 95 (a protein regulated the spine synaptic densities) across the F3 juvenile male mice (Wolstenholme et al., 2019). In their study, numeric spines densities were decreased in those BPA-exposed male offspring, which is consistent with our results that maternal BPA exposure (all doses) decreased the CA1 dendritic spine densities across both F1 and F2 male generations.

Table 2 Comet percentages and arbitrary units of brain cells in F1 and F2 mice after different BPA treatments(N = 16). Group (μg/kg/day)

F1

F2

0 0.5 50 5000 0 0.5 50 5000

The numbers of comet cells in different grades(n = 800) Gd0

Gd1

Gd2

Gd3

Gd4

606 539 610 609 584 562 597 612

157 181 153 148 163 182 168 150

36 77 37 40 47 51 31 35

1 3 0 3 6 2 4 3

0 0 0 0 0 0 0 0

The comet percentage (%)

Arbitrary units

24.3 32.6 23.8 23.9 27.0 29.5 25.4 23.5

0.29 0.43 0.28 0.30 0.34 0.36 0.30 0.29

There were 16 mice for each group in F1 and F2, respectively.

treatment of BPA could cause significant alterations to 5-HT, DA, GABA and Glu in female, but not in male offspring (Table 3). Notably, significantly increased GABA levels and significantly decreased Glu/GABA ratios were observed in both the medium- and high-exposed male and female mice of F1 (p < 0.05 or p < 0.01). Maternal exposure to medium dosage of BPA also remarkably altered the 5-HT levels in the hippocampi of F1 male mice (Table 3).

4.2. The sex-dependent effects of BPA exposure Neuron numbers in hippocampal subregions across both the F1 and F2 mice were decreased along with the neuron morphological alterations resulting from maternal BPA exposure in this study. It is critical for development of the nervous system because a majority of neurons involved in hippocampus formation are generated prenatally, and the generation speed of granule cells in DG during the adult period is very slow (Kaplan and Bell, 1983). Previous study showed that high-dose BPA (10 mg/kg/day) exposure during pregnancy (F0) significantly decreased the cells of F2 female mice in the granular cell layer of hippocampal DG, suggesting impaired hippocampal DG neurogenesis (Jang et al., 2012), which reflects our results as well. In this study, high (5000 μg/kg/day) and medium levels (50 μg/kg/day) of maternal BPA exposure significantly decreased neuron quantities of hippocampal DG across both the F1 and F2 female and male mice (Fig. 2). Interestingly, our results differ from other studies in that even environmental BPA exposure (0.5 μg/kg/day) could decrease the neuron quantities of hippocampal DG across the F2 male mice (Fig. 2), which raises concerns. Besides in the hippocampal DG region, we also observed a significant decrease of neuron quantities in CA1 region of F2 male offspring induced by low-dose BPA exposure. These findings provide robust evidence that even environmental BPA exposure during early neuron development can cause toxic effects on brain histology and this influence could carry over to future generations. However, the potential mechanism is not yet clear. Some studies have suggested an explanation with reference to altered epigenetic mechanisms, such as DNA methylation (Beri et al., 2007; Jang et al., 2012) and histone modifications (Camacho and Allard, 2018). Another notable point is that the neurotoxic effects induced by maternal BPA exposure on offspring generally were observed in a sexdependent manner. Comprehensively, BPA caused more significant neurotoxic effects on male than female mice. For example, the Y-maze test showed that F1 male mice without BPA exposure exhibited better performance in the spatial learning and memory task than the female mice. However, after maternal BPA exposure, this sexually dimorphic behavior was disrupted and the exposed F1 male mice exhibited worse spatial learning and memory abilities than the females. The phenomenon was distinctive between mice from the low-exposed group (Table 1), suggesting male-dependent brain DNA damage (Fig. 1). Additionally, the dendritic spine densities in hippocampal CA1 of both the F1 and F2 male offspring generally seemed to be more vulnerable to BPA exposure compared with those in female offspring (Fig. 3). These

4. Discussion 4.1. The multigenerational effects of maternal BPA exposure on neuron development To investigate whether maternal exposure to low-dosage BPA can exhibit multi- or transgenerational effects on central nervous system and learning and memory abilities, the influence of BPA exposure on the brain DNA damage and the hippocampal morphology in the F1 and F2 offspring were studied initially. Then the alteration of neurotransmitter levels and learning and memory abilities induced by BPA exposure were studied to test the hypothesis that maternal BPA exposure exhibit genotoxicity across generations by involving the alteration of hippocampus morphologies and neurotransmitter levels. In this study, comet assay results showed that maternal low- and medium-dose BPA exposure (0.5 and 50 μg/kg/day) could lead to significant DNA damage in the brain cells of F1 male and female mice (Fig. 1), seemingly demonstrating its genotoxicity. However, such DNA damage was not observed in F2 mice. We deduced that the damage might be repaired through epigenetic mechanisms in F2 as BPA promoted the DNA repair capacity. For example, Gensan and Keating found that BPA exposure could increase the expressions of several DNA repair genes in germ cells (Ganesan and Keating, 2016). In fact, several previous studies of multi- or transgenerational effects of BPA on central nervous system and neurobehaviors are controversial. Wolstenholme et al. and Jang et al. found that maternal BPA exposure caused deficits in social recognition and passive avoidance learning and memory across F2 and F3 generations (Jang et al., 2012; Wolstenholme et al., 2013, 2019), suggesting multi- or transgenerational effects of BPA exposure. While Xin et al. found that depressive-like behavior was only limited to the exposed F1 male generation after maternal BPA exposure (Xin et al., 2018). In this study, the results of the Y-maze test showed that impaired spatial learning and memory ability was limited to the exposed F1 male mice. We did not observe the altered behavior in F1 female mice and F2 mice (Table 1). However, the results of hippocampal analysis seem to suggest that maternal BPA exposure could exert toxic effects on nervous systems across generations, including decreased neuron quantities and dendritic spine densities in hippocampi of F1 and F2 mice, particularly in male mice. This neurotoxic evidence is substantial and reliable because neurons in the hippocampus are important parts of the nervous system responsible for 6

Toxicology 432 (2020) 152393

H. Zhang, et al.

Fig. 3. Effects of BPA exposure on hippocampal dendritic arbor development for offspring mice. (A) Representative images of dendritic tracings of CA1 pyramidal neurons of F1 male mice from the control, BPA 0.5, BPA 50 and BPA 5000 groups. (B, C) CA1 dendritic intersections and total length of F1 male mice decreased significantly in BPA exposed groups. Representative images of dendritic shaft with spines of CA1 pyramidal neurons of F1(D) and F2 (E) mice from the control, BPA-0.5, BPA-50 and BPA 5000 groups. (F) Perinatal BPA exposure significantly decreased the CA1 densities of F1 and F2 male mice. There were 6 male mice and 6 female mice in each group. (*p < 0.05, **p < 0.01, compared with the control group).

results suggest that the development of learning and memory processes in males are more vulnerable to maternal BPA exposure. Environmental BPA levels can induce long-lasting neurotoxicities in the male offspring. Previous studies also showed that impaired learning and memory are

linked to BPA in males no matter what differences in species, age at/ and route of exposure, or BPA dose (Elsworth et al., 2015; Jain et al., 2011; Jašarević et al., 2011, 2013; Kumar and Thakur, 2014; Li et al., 2018; Xu et al., 2011b, 2014; Zhou et al., 2017). Considering estrogens 7

Toxicology 432 (2020) 152393

H. Zhang, et al.

Table 3 Concentrations of neurotransmitters in hippocampus of F1 offspring after maternal BPA exposure. Gender

Group (μg/kg/day)

Glu

GABA

Glu/GABA

ACh

5-HT

DA

Male (μg/g)

Control (N = 12) BPA 0.5 (N = 11) BPA 50 (N = 10) BPA 5000 (N = 9) Control (N = 11) BPA 0.5 (N = 11) BPA 50 (N = 12) BPA 5000 (N = 10)

1282 ± 85.1 1317 ± 160 1592 ± 127 1287 ± 151 1151 ± 63.5 1408 ± 77.1* 1459 ± 111* 1702 ± 216*

176 ± 16.6 203 ± 29.2 255 ± 14.7** 264 ± 26.7* 165 ± 12.0 232 ± 15.4** 241 ± 15.7** 257 ± 17.8**

7.49 ± 0.280 6.75 ± 0.338 6.22 ± 0.292** 5.23 ± 0.642** 7.15 ± 0.328 6.26 ± 0.424 5.81 ± 0.156** 5.74 ± 0.447*

0.722 ± 0.102 0.667 ± 0.116 1.03 ± 0.267 0.805 ± 0.193 0.590 ± 0.102 0.789 ± 0.160 0.935 ± 0.207 1.17 ± 0.311

0.639 ± 0.0406 0.726 ± 0.101 1.13 ± 0.0567** 0.818 ± 0.0861 0.632 ± 0.0622 0.932 ± 0.121* 1.07 ± 0.0474** 1.23 ± 0.123**

1.09 ± 0.0989 0.986 ± 0.141 1.37 ± 0.0780 1.13 ± 0.113 0.884 ± 0.0770 1.30 ± 0.191* 1.23 ± 0.147* 1.95 ± 0.283**

Female (μg/g)

*or ** indicates that there was a significant difference between the control and exposed groups (p < 0.05 or p < 0.01).

are a class of hormones involved in brain neurodevelopment. The relationship between different disruptions in the binding activities of estrogen receptors and BPA exposure may explain sex-different neurotoxicities (McCarthy, 2008). It is worth to note that maternal BPA exposure led to variations in hippocampal neurotransmitter levels in F1 offspring (Table 3). The significantly decreased ratios of Glu/GABA observed in both the medium- and high-exposed mice were accompanied with the impaired hippocampal DG neurogenesis mentioned above. Neurotransmitter systems have been reported to be involved in learning and memory (Myhrer, 2003). Balance between Glu and GABA is vital for normal brain functions as they are excitatory and inhibitory neurotransmitters in the central nervous system, respectively (Car and Wiśniewski, 1998). Our findings suggest that the disrupted balance between Glu and GABA is correlated with the neurotoxicities in male offspring’s developing brains because the exposed male mice with disrupted Glu/GABA ratios also inhibited learning abilities. Additionally, low-dose maternal BPA exposure could alter the hippocampal Glu, GABA, 5-HT and DA levels in F1 female offspring, though with no significant changes in learning and memory ability. In a word, though maternal BPA exposure could exert toxic effects on hippocampal neurons across mice generations, its genotoxic evidences need more and in-depth studies. All our results could deduce that BPA is primarily acting as an endocrine disruptor to affect brain development by impairing of the balance of neurotransmitter levels and influencing neuron morphology. Certainly, we need more works to support our synthesis. In the future, estrogen receptors (ERs) expression, neurotransmitter levels and their receptor’s expression, expression of key proteins in synaptic remodeling of neurons as well as key genes expression in BPA-induced signaling pathways will be thoroughly investigated in our lab to explore these sex and level discrepancies.

Though 50 μg/kg intakes of BPA per day does not significantly increase occurrences of breast or prostate cancer (U.S. EPA, 1988), evidences of negative health effects induced by environmental BPA exposure emerge gradually, which makes it necessary to re-adjust the standard. A vulnerable organ or sensitive health effect outcome is more suitable to assess the toxicity of a pollutant than a standard based on its carcinogenic or teratogenic effects. 5. Conclusions In summary, maternal exposure to BPA, even to an environmental dose, impaired the learning and memory ability for F1 male offspring mice. Moreover, maternal BPA exposure decreased neuron quantities and dendritic spine density in hippocampi across generations. However, significant brain DNA damage was only limited to F1 offspring (i.e. the low-exposed male mice and medium-exposed female mice). The neurotoxic effects induced by maternal BPA exposure on offspring generally occurred in a sex-dependent manner. Maternal BPA exposure also led to variations in hippocampal neurotransmitter levels in F1 offspring. Overall, this study highlights the susceptibility of neurodevelopment in crucial periods to BPA exposure, and suggests bringing more attention to the neurotoxic effects of maternal BPA exposure on offspring. Funding This research was supported by grants from the National Natural Science Foundation of China (No. 21777048, 41731279 and No. 21477041). Declaration of Competing Interest The authors declare no conflict of interest.

4.3. The necessity of reassessing BPA safety by using a sensitive health outcome

Appendix A. Supplementary data

BPA exposure during prenatal, postnatal and adolescent periods has well-documented neurotoxic effects on the developing brain and behaviors (Anderson et al., 2013; Cox et al., 2010; Gore et al., 2018; Mhaouty-Kodja et al., 2018; Stein et al., 2015; Tewar et al., 2016; Weinstein et al., 2013; Zhou et al., 2017). Our and previous animal studies all confirmed by different means and methods that maternal BPA exposure has multigenerational effects on neurons or brain development (Drobná et al., 2017; Jang et al., 2012; Wolstenholme et al., 2012, 2013, 2019). This suggests that in addition to reducing BPA intake in infants and young children, mothers should also try to reduce BPA intake during pre-pregnancy, pregnancy and lactation. Literatures on the adverse health effects caused by environmental BPA exposure are increasing rapidly (Li et al., 2018; Xin et al., 2018; Zhou et al., 2017). More and more studies suggest that we should reassess the safety of BPA intake and redefine the intake standard of BPA (European Food Safety Authority (EFSA, 2015). The current safety standards for BPA are set according to teratogenic, carcinogenic and mutagenic effects.

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.tox.2020.152393. References Anderson, O.S., Peterson, K.E., Sanchez, B.N., Zhang, Z., Mancuso, P., Dolinoy, D.C., 2013. Perinatal bisphenol A exposure promotes hyperactivity, lean body composition, and hormonal responses across the murine life course. FASEB J . 27 (4), 1784–1792. Audebert, M., Dolo, L., Perdu, E., Cravedi, J.P., Zalko, D., 2011. Use of the γH2AX assay for assessing the genotoxicity of bisphenol A and bisphenol F in human cell lines. Arch.Toxicol. 85 (11), 1463–1473. Bannerman, D.M., Sprengel, R., Sanderson, D.J., McHugh, S.B., Rawlins, J.N.P., Monyer, H., Seeburg, P.H., 2014. Hippocampal synaptic plasticity, spatial memory and anxiety. Nat. Rev. Neurosci. 15 (3), 181. Battal, D., Cok, I., Unlusayin, I., Aktas, A., Tunctan, B., 2014. Determination of urinary levels of Bisphenol A in a Turkish population. Environ. Monit. Assess. 186 (12), 8443–8452. Beatty, W.W., 1979. Gonadal hormones and sex differences in nonreproductive behaviors in rodents: organizational and activational influences. Horm. Behav. 12 (2), 112–163.

8

Toxicology 432 (2020) 152393

H. Zhang, et al. Beri, S., Tonna, N., Menozzi, G., Bonaglia, M.C., Sala, C., Giorda, R., 2007. DNA methylation regulates tissue-specific expression of Shank3. J. Neurochem. 101 (5), 1380–1391. Bernal, A.J., Jirtle, R.L., 2010. Epigenomic disruption: the effects of early developmental exposures. Birth Defects Res A Clin Mol Teratol. 88 (10), 938–944. Bonthuis, P.J., Cox, K.H., Searcy, B.T., Kumar, P., Tobet, S., Rissman, E.F., 2010. Of mice and rats: key species variations in the sexual differentiation of brain and behavior. Front. Neuroendocrinol. 31 (3), 341–358. Camacho, J., Allard, P., 2018. Histone modifications: epigenetic mediators of environmental exposure memory. Epigenet Insights. 11, 2516865718803641. Car, H., Wiśniewski, K., 1998. Similarities and interactions between GABAergic and glutaminergic systems. Rocz Akad Med Bialymst. 43, 5–26. Chouhan, S., Yadav, S.K., Prakash, J., Singh, S.P., 2014. Effect of Bisphenol A on human health and its degradation by microorganisms: a review. Ann. Microbiol. 64 (1), 13–21. Cox, K.H., Gatewood, J.D., Howeth, C., Rissman, E.F., 2010. Gestational exposure to bisphenol A and cross-fostering affect behaviors in juvenile mice. Horm. Behav. 58 (5), 754–761. Cravedi, J.P., Zalko, D., Savouret, J.F., Menuet, A., Jegou, B., 2007. The concept of endocrine disruption and human health. Med. Sci. 23 (2), 198–204. Drobná, Z., Henriksen, A.D., Wolstenholme, J.T., Montiel, C., Lambeth, P.S., Shang, S., Harris, E.P., Zhou, C., Flaws, J.A., Adli, M., 2017. Transgenerational effects of bisphenol A on gene expression and DNA methylation of imprinted genes in brain. Endocrinology 159 (1), 132–144. Elsworth, J.D., Jentsch, J.D., Groman, S.M., Roth, R.H., Redmond Jr., E.D., Leranth, C., 2015. Low circulating levels of bisphenol‐A induce cognitive deficits and loss of asymmetric spine synapses in dorsolateral prefrontal cortex and hippocampus of adult male monkeys. J. Comp. Neurol. 523 (8), 1248–1257. European Food Safety Authority (EFSA). Bisphenol A. Available at http://www. efsa. europa.eu/en/topics/topic/bisphenol, accessed on Janurary 2015. Fan, R., Zeng, B., Liu, X., Chen, C., Zhuang, Q., Wang, Y., Hu, M., Lv, Y., Li, J., Zhou, Y., 2015. Levels of bisphenol-A in different paper products in Guangzhou, China, and assessment of human exposure via dermal contact. Environ.Sci: Processes & Impacts. 17 (3), 667–673. Food and Drug Administration (FDA), 2012. Indirect Food Additives 77. Federal Register. Available at http://www.gpo.gov/fdsys/pkg/FR-2012-0717/pdf/2012-17366.pdf, accessed on July. Ganesan, S., Keating, A.F., 2016. Bisphenol A-induced ovotoxicity involves DNA damage induction to which the ovary mounts a protective response indicated by increased expression of proteins involved in DNA repair and xenobiotic biotransformation. Toxicol. Sci. 152, 169–180. Geens, T., Goeyens, L., Covaci, A., 2011. Are potential sources for human exposure to bisphenol-A overlooked? Int. J. Hyg. Environ. Health 214 (5), 339–347. Gore, A.C., Krishnan, K., Reilly, M.P., 2018. Endocrine-disrupting chemicals: effects on neuroendocrine systems and the neurobiology of social behavior. Horm. Behav. 111, 7–22. Government of Canada, 2010. Order Amending Schedule I to the Hazardous Products Act (Bisphenol A), Part II. 144, p. 7., accessed on March. . Ikezuki, Y., Tsutsumi, O., Takai, Y., Kamei, Y., Taketani, Y., 2002. Determination of bisphenol A concentrations in human biological fluids reveals significant early prenatal exposure. Hum. Reprod. 17 (11), 2839–2841. Iso, T., Watanabe, T., Iwamoto, T., Shimamoto, A., Furuichi, Y., 2006. DNA damage caused by bisphenol A and estradiol through estrogenic activity. BiolPharm Bulletin 29 (2), 206–210. Jain, S., Kumar, C.M., Suranagi, U.D., Mediratta, P.K., 2011. Protective effect of N-acetylcysteine on bisphenol A-induced cognitive dysfunction and oxidative stress in rats. Food Chem. Toxicol. 49 (6), 1404–1409. Jang, Y.J., Park, H.R., Kim, T.H., Yang, W.J., Lee, J.J., Choi, S.Y., Oh, S.B., Lee, E., Park, J.H., Kim, H.P., 2012. High dose bisphenol A impairs hippocampal neurogenesis in female mice across generations. Toxicol. 296 (1–3), 73–82. Jašarević, E., Sieli, P.T., Twellman, E.E., Welsh, T.H., Schachtman, T.R., Roberts, R.M., Geary, D.C., Rosenfeld, C.S., 2011. Disruption of adult expression of sexually selected traits by developmental exposure to bisphenol. A. Proc. Natl. Acad. Sci. U. S. A. 108 (28), 11715–11720. Jašarević, E., Williams, S.A., Vandas, G.M., Ellersieck, M.R., Liao, C., Kannan, K., Roberts, R.M., Geary, D.C., Rosenfeld, C.S., 2013. Sex and dose-dependent effects of developmental exposure to bisphenol A on anxiety and spatial learning in deer mice (Peromyscus maniculatus bairdii) offspring. Horm. Behav. 63 (1), 180–189. Kaplan, M., Bell, D., 1983. Neuronal proliferation in the 9-month-old rodent—radioautographic study of granule cells in the hippocampus. Exp. Brain Res. 52 (1), 1–5. Kimura, E., Matsuyoshi, C., Miyazaki, W., Benner, S., Hosokawa, M., Yokoyama, K., Kakeyama, M., Tohyama, C., 2016. Prenatal exposure to bisphenol A impacts neuronal morphology in the hippocampal CA1 region in developing and aged mice. Arch. Toxicol. 90 (3), 691–700. Kuang, H., Zhang, H., Tian, J., Luo, Y., Liu, S., Pang, Q., Fan, R., 2019. Simultaneous determination of 5 neurotransmitters in neonatal rat hippocampus by adding Vitamin C coupled with isotope dilution-UPLC-MS/MS method. Chinese J. Chromatogr. 37, 404–411. Kumar, D., Thakur, M.K., 2014. Perinatal exposure to bisphenol-A impairs spatial memory through upregulation of neurexin1 and neuroligin3 expression in male mouse brain. PLoS One 9 (10), e110482. Li, F., Yan, C.Q., Lin, L.T., Li, H., Zeng, X.H., Liu, Y., Du, S.Q., Zhu, W., Liu, C.Z., 2015. Acupuncture attenuates cognitive deficits and increases pyramidal neuron number in hippocampal CA1 area of vascular dementia rats. BMC Complement. Altern. Med. 15 (1), 133.

Li, Y., Zhang, H., Kuang, H., Fan, R., Cha, C., Li, G., Luo, Z., Pang, Q., 2018. Relationship between bisphenol A exposure and attention-deficit/hyperactivity disorder: a casecontrol study for primary school children in Guangzhou, China. Environ. Pollut. 235, 141–149. Liu, Z.H., Ding, J.J., Yang, Q.Q., Song, H.Z., Chen, X.T., Xu, Y., Xiao, G.R., Wang, H.L., 2016. Early developmental bisphenol-A exposure sex-independently impairs spatial memory by remodeling hippocampal dendritic architecture and synaptic transmission in rats. Sci. Rep. 6, 32492. Luo, G., Wei, R., Niu, R., Wang, C., Wang, J., 2013. Pubertal exposure to Bisphenol A increases anxiety-like behavior and decreases acetylcholinesterase activity of hippocampus in adult male mice. Food Chem. Toxicol. 60 (10), 177–180. McCarthy, M.M., 2008. Estradiol and the developing brain. Physiol. Rev. 88 (1), 91–134. Meeker, J.D., Sathyanarayana, S., Swan, S.H., 2009. Phthalates and other additives in plastics: human exposure and associated health outcomes. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 364 (1526), 2097–2113. Mhaouty-Kodja, S., Belzunces, L.P., Canivenc, M.C., Schroeder, H., Chevrier, C., Pasquier, E., 2018. Impairment of learning and memory performances induced by BPA: evidences from the literature of a MoA mediated through an ED. Mol. Cell. Endocrinol. 475, 54–73. Michałowicz, J., 2014. Bisphenol A–sources, toxicity and biotransformation. Environ. Toxicol. Pharmacol. 37, 738–758. Moser, E.I., Roudi, Y., Witter, M.P., Kentros, C., Bonhoeffer, T., Moser, M.B., 2014. Grid cells and cortical representation. Nat. Rev. Neurosci. 15 (7), 466. Munguia-Lopez, E.M., Soto-Valdez, H., 2001. Effect of heat processing and storage time on migration of bisphenol A (BPA) and bisphenol A− diglycidyl ether (BADGE) to aqueous food simulant from Mexican can coatings. J. Agric. Food Chem. 49 (8), 3666–3671. Myhrer, T., 2003. Neurotransmitter systems involved in learning and memory in the rat: a meta-analysis based on studies of four behavioral tasks. Brain Res. Rev. 41 (2–3), 268–287. Rice, D., Barone Jr., S., 2000. Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ. Health Perspect. 108 (suppl3), 511–533. Richter, C.A., Birnbaum, L.S., Farabollini, F., Newbold, R.R., Rubin, B.S., Talsness, C.E., Vandenbergh, J.G., Walser-Kuntz, D.R., vom Saal, F.S., 2007. In vivo effects of bisphenol A in laboratory rodent studies. Reprod. Toxicol. 24 (2), 199–224. Sasaki, N., Okuda, K., Kato, T., Kakishima, H., Okuma, H., Abe, K., Tachino, H., Tuchida, K., Kubono, K., 2005. Salivary bisphenol-A levels detected by ELISA after restoration with composite resin. J. Mater. Sci. Mater. Med. 16 (4), 297–300. Stein, T.P., Schluter, M.D., Steer, R.A., Guo, L., Ming, X., 2015. Bisphenol A exposure in children with autism spectrum disorders. Autism Res. 8 (3), 272–283. Sun, Y., Nakashima, M.N., Takahashi, M., Kuroda, N., Nakashima, K., 2002. Determination of bisphenol A in rat brain by microdialysis and column switching high‐performance liquid chromatography with fluorescence detection. Biomed. Chromatogr. 16 (5), 319–326. Tewar, S., Auinger, P., Braun, J.M., Lanphear, B., Yolton, K., Epstein, J.N., Ehrlich, S., Froehlich, T.E., 2016. Association of bisphenol A exposure and attention-deficit/ hyperactivity disorder in a national sample of US children. Environ. Res. 150, 112–118. U.S. EPA (U.S. Environmental Protection Agency), 1988. Bisphenol A (CASRN 80-05-7). Available. http://www.epa.gov/iris/subst/0356.htm! (Accessed 25 November2007). . U.S. EPA (United States Environmental Protection Agency), 2012. Exposure Factors. http://www.epa.gov/oppt/exposure/presentations/efast/usepa1997efh.pdf. Ulutaş, O.K., Yıldız, N., Durmaz, E., Ahbab, M.A., Barlas, N., Çok, İ., 2011. An in vivo assessment of the genotoxic potential of bisphenol A and 4-tert-octylphenol in rats. Arch. Toxicol. 85, 995–1001. Vandenberg, L.N., Hauser, R., Marcus, M., Olea, N., Welshons, W.V., 2007. Human exposure to bisphenol A (BPA). Reprod. Toxicol. 24 (2), 139–177. Vandenberg, L.N., Chahoud, I., Heindel, J.J., Padmanabhan, V., Paumgartten, F.J., Schoenfelder, G., 2010. Urinary, circulating, and tissue biomonitoring studies indicate widespread exposure to bisphenol A. Environ Health Persp. 118 (8), 1055–1070. Vom Saal, F.S., Akingbemi, B.T., Belcher, S.M., Birnbaum, L.S., Crain, D.A., Eriksen, M., Farabollini, F., Guillette, Jr L.J., Hauser, R., Heindel, J.J., 2007. Chapel Hill bisphenol A expert panel consensus statement: integration of mechanisms, effects in animals and potential to impact human health at current levels of exposure. Reprod toxicol (Elmsford, NY) 24 (2), 131. Weinstein, S.D., Villafane, J.J., Juliano, N., Bowman, R.E., 2013. Adolescent exposure to Bisphenol-A increases anxiety and sucrose preference but impairs spatial memory in rats independent of sex. Brain Res. 1529 (13), 56–65. Wolstenholme, J.T., Edwards, M., Shetty, S.R., Gatewood, J.D., Taylor, J.A., Rissman, E.F., Connelly, J.J., 2012. Gestational exposure to bisphenol a produces transgenerational changes in behaviors and gene expression. Endocrinol 153 (8), 3828–3838. Wolstenholme, J.T., Goldsby, J.A., Rissman, E.F., 2013. Transgenerational effects of prenatal bisphenol A on social recognition. Horm. Behav. 64 (5), 833–839. Wolstenholme, J.T., Drobn, Z., Henriksen, A.D., Goldsby, J.A., Stevenson, R., Irvin, J.W., Flaws, J.A., Rissman, E.F., 2019. Transgenerational Bisphenol A causes deficits in social recognition and alters post-synaptic density genes in mice. Endocrinol 160 (8), 1854–1867. Xin, F., Jiang, L., Liu, X., Geng, C., Wang, W., Zhong, L., Yang, G., Chen, M., 2014. Bisphenol A induces oxidative stress-associated DNA damage in INS-1 cells. Mutat Res /Genet Toxicol Environ Mutag 769, 29–33. Xin, F., Fischer, E., Krapp, C., Krizman, E.N., Lan, Y., Mesaros, C., Snyder, N.W., Bansal, A., Robinson, M.B., Simmons, R.A., 2018. Mice exposed to bisphenol A exhibit

9

Toxicology 432 (2020) 152393

H. Zhang, et al. depressive-like behavior with neurotransmitter and neuroactive steroid dysfunction. Horm. Behav. 102, 93–104. Xu, X., Li, T., Luo, Q., Hong, X., Xie, L., Tian, D., 2011a. Bisphenol-A rapidly enhanced passive avoidance memory and phosphorylation of NMDA receptor subunits in hippocampus of young rats. Toxicol. Appl. Pharmacol. 255 (2), 221–228. Xu, X., Tian, D., Hong, X., Chen, L., Xie, L., 2011b. Sex-specific influence of exposure to bisphenol-A between adolescence and young adulthood on mouse behaviors. Neuropharmacol. 61 (4), 565–573. Xu, X.B., He, Y., Song, C., Ke, X., Fan, S.J., Peng, W.J., Tan, R., Kawata, M., Matsuda, K.I.,

Pan, B.X., 2014. Bisphenol A regulates the estrogen receptor alpha signaling in developing hippocampus of male rats through estrogen receptor. Hippocampus. 24 (12), 1570–1580. Zaqout, S., Kaindl, A.M., 2016. Golgi-cox staining step by step. Front. Neuroanat. 10, 38. Zhou, Y., Wang, Z., Xia, M., Zhuang, S., Gong, X., Pan, J., Li, C., Fan, R., Pang, Q., Lu, S., 2017. Neurotoxicity of low bisphenol A (BPA) exposure for young male mice: Implications for children exposed to environmental levels of BPA. Environ Pollut 22, 40–48.

10