Parental co-exposure to bisphenol A and nano-TiO2 causes thyroid endocrine disruption and developmental neurotoxicity in zebrafish offspring

Science of the Total Environment 650 (2019) 557–565

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Parental co-exposure to bisphenol A and nano-TiO2 causes thyroid endocrine disruption and developmental neurotoxicity in zebrafish offspring Yongyong Guo a ,1 , Lianguo Chen a ,1 , Juan Wu a , Jianghuan Hua a , Lihua Yang a , Qiangwei Wang b , Wei Zhang c , Jae-Seong Lee d , Bingsheng Zhou a ,⁎ a

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, China c State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, China d Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 16419, South Korea b

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Chronic co-exposure to BPA and nanoTiO2 to a zebrafish model was conducted. • Co-exposure increased bioaccumulation and transfer of BPA and n-TiO2 to offspring. • Thyroid hormones were further decreased by co-exposure in F0 and F1 generations. • Developmental neurotoxicity was observed in offspring larvae from exposed parents. • Antagonistic and synergistic interactions depended on the BPA concentration.

a r t i c l e

i n f o

Article history: Received 21 June 2018 Received in revised form 31 August 2018 Accepted 1 September 2018 Available online 03 September 2018 Editor: Jay Gan Keywords: Bisphenol A n-TiO2 Chronic co-exposure Maternal transfer Developmental neurotoxicity and thyroid disruption Zebrafish

a b s t r a c t The coexistence of organic toxicants and nanoparticles in the environment influences pollutant bioavailability and toxicity. Using chronic co-exposure to an adult zebrafish model, this study investigated the transfer kinetics and transgenerational effects of bisphenol A (BPA) and titanium dioxide nanoparticles (n-TiO2) exposure in F1 offspring. When single and combined exposure to BPA (0, 2, and 20 μg/L) and n-TiO2 (100 μg/L) were compared, combined exposure was found to reciprocally facilitate bioaccumulation in adult fish while enhancing maternal transfer to offspring. Thyroid endocrine disruption and developmental neurotoxicity were observed in larval offspring by parental exposure to BPA alone or in combination with n-TiO2. Exposure to 20 μg/L BPA significantly decreased the thyroxine (T4) concentration in adult plasma, leading to less transfer into the eggs. The presence of 20 μg/L BPA with n-TiO2 further decreased the level of T4 compared to BPA exposure alone. Additionally, offspring larvae derived from exposed parents exhibited lethargic swimming behavior. Overall, this study examined the interactions of BPA and n-TiO2 with regard to their bioaccumulation, maternal transfer, and developmental effects, which highlighted that co-exposure dynamics are important and need to be considered for accurate environmental risk assessment. © 2018 Elsevier B.V. All rights reserved.

⁎ Corresponding author. E-mail address: [email protected] (B. Zhou). 1 Yongyong Guo and Lianguo Chen equally contributed to this work.

https://doi.org/10.1016/j.scitotenv.2018.09.007 0048-9697/© 2018 Elsevier B.V. All rights reserved.

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Y. Guo et al. / Science of the Total Environment 650 (2019) 557–565

1. Introduction Bisphenol A (BPA) is widely used in the manufacture of a myriad of commercial products, such as dental sealants, food packaging, water containers, baby bottles, and medical equipment (Hoekstra and Simoneau, 2013). Because of its persistent nature, contamination by BPA has been detected in various environmental matrices. Specifically in the water samples from several rivers in China, BPA accumulation has been detected to be 4 μg/L (Huang et al., 2012). In contaminated rivers, the concentration of BPA has been recorded as high as 28 μg/L (Heisterkamp et al., 2004). This ubiquitous presence exposes animals and humans to the continuous hazards associated with BPA (Flint et al., 2012; Gu et al., 2016; Vandenberg et al., 2013). Previous investigations into BPA have mainly focused on the identification of its estrogenic activities and reproductive impairing potencies (Bhandari et al., 2015; Ding et al., 2017; Michalowicz, 2014). However, disruption of the thyroid endocrine system is another side-effect of BPA exposure, which needs to be further investigated. The direct effect of BPA exposure on zebrafish thyroid follicular cells has been reported both in vitro and in vivo (Gentilcore et al., 2013). The binding of BPA to thyroid receptors also disrupts regulative mechanisms along the hypothalamus-pituitary-thyroid (HPT) axis, resulting in imbalanced thyroid hormone (TH) homeostasis (Lee et al., 2017; Zhang et al., 2018; Zoeller et al., 2005). BPA is also reported to inhibit sodium/iodide symporter (NIS), thereby decreasing the uptake of iodide in the thyroid (Wu et al., 2016). In addition to endocrine disruption, exposure to BPA during early development also induces neurotoxic effects by affecting brain organization and behavioral outcomes (e.g., anxiolytic behavior with cognitive deficits) in rodent models (Wolstenholme et al., 2012). Epidemiological studies have recently demonstrated that exposure to BPA during pregnancy is linked to increased risk of anxiety and depression (Harley et al., 2013; Perera et al., 2012) and the development of autism spectrum disorders (Stein et al., 2015) later in life. As a common engineered nanomaterial, nanoscale titanium dioxide (n-TiO2) is one of the most highly produced in the world (Robichaud et al., 2009), and is extensively used in industrial and commercial products, including personal care products, surface coatings, paints, sunscreens, and food (Weir et al., 2012). Additionally, n-TiO2 is also used in photocatalytic devices to degrade many organic contaminants and achieve environmental remediation (Chong et al., 2010). Therefore, n-TiO2 is inevitably released into the environment. Modeling studies predict that the environmental concentrations of n-TiO2 in water are in the order of ppb (Gottschalk et al., 2011; Mueller and Nowack, 2008). This raises growing concerns about its potential to elicit adverse health and environmental effects. The sub-lethal effects of n-TiO 2 exposure on aquatic life are reported to include oxidative stress and gill pathology reactions (Federici et al., 2007), retarded oogenesis, and impaired reproduction (Wang et al., 2011). Furthermore, 45-day exposure of zebrafish to n-TiO2 particles at low doses (5, 10, 20, and 40 μg/L) results in brain injuries, neurochemical composition alterations, and spatial recognition memory impairments (Sheng et al., 2016). Due to the high surface reactivity and large surface area of n-TiO2, they have a high tendency to amalgamate with other pollutants in natural environments and exhibit interactive effects thereafter, which may alter their innate environmental behavior, consequently changing toxicological effects (Naasz et al., 2018; Tan et al., 2012). Our previous studies have demonstrated that interaction between n-TiO2 and BPA, either synergistic or antagonistic, results in zebrafish sex endocrine disruption and reproductive impairment (Fang et al., 2016), and gut microbiota dysbiosis (Chen et al., 2018a). Given the fact that n-TiO2 acts as a carrier of BPA to enhance its bioconcentration in zebrafish brain and gonads (Fang et al., 2016), in the present study, we focused on parental longterm co-exposure of n-TiO2 and BPA and examined the resulting transfer of these compounds to offspring. Effects of parental co-exposure on

larval offspring (i.e., thyroid endocrine disruption and developmental neurotoxicity) were investigated to determine possible changes in the innate toxic effects of BPA and n-TiO2. 2. Materials and methods 2.1. Chemicals BPA and tricaine mesylate (MS-222) were obtained from Sigma Aldrich (St. Louis, MO, USA). BPA was dissolved in dimethyl sulfoxide (DMSO) and stored at 4 °C. n-TiO2 was purchased from Hangzhou Wan Jing New Material Company (purity N 99.9%; CAS 13463-67-7; China). The average diameter of the nano-TiO2 nanoparticles was 5 nm according to the manufacturer's description. Stock solutions of n-TiO2 (1 mg/mL) were prepared by dispersing in ultrapure water (Millipore, Billerica, MA, USA) with sonication (50 W/L, 40 kHz) for 20 min. Test n-TiO2 solutions were prepared immediately by diluting the stock solutions with fresh charcoal-filtered water. The properties of the n-TiO2 used in the present study have been characterized previously (Wang et al., 2014). All other chemicals used in this study were of analytical grade and trace metal analysis grade for TiO2 analysis. 2.2. Zebrafish maintenance and experiment design All experiments were performed using wild type AB strain adult zebrafish (90 days post fertilization, dpf) in a semi-static system containing charcoal-filtered and fully-aerated tap water under a constant ambient environment (temperature: 28 ± 0.5 °C; light:dark cycle of 14 h:10 h). The zebrafish were exposed to gradient concentrations of BPA (0, 2, and 20 μg/L) alone or in combination with n-TiO2 (100 μg/L). BPA and nanoparticles were spiked into the co-exposure media separately. Because previous monitoring reports the level of BPA in the aquatic environment to be up to 28 μg/L (Heisterkamp et al., 2004), the BPA exposure concentrations used in the present study reflected its actual environmental concentrations. The exposure concentration of nano-TiO2 at 100 μg/L was also environmentally relevant given that the predicted environmental concentration is 0.7–16 μg/L in the waters of Switzerland (Mueller and Nowack, 2008). At such a concentration, TiO2 nanoparticles have been shown to significantly compromise organismal health after chronic exposure (Chen et al., 2018a; Fang et al., 2016). Three identical replicate tanks were used for each exposure group, with each tank containing 12 males and 12 females in 20 L of exposure medium. During the experimental period, the exposure solution was renewed daily, and both the control and exposure groups received 0.005% DMSO (v/v). The fish were fed twice every day with pellet food (Zeigler Brothers, Gardners, PA, USA) and freshly hatched Artemia nauplii. After four months of exposure, both the control and exposed fish were paired in clean water and the eggs were immediately collected for chemical analysis of the BPA and n-TiO2 content and for thyroid hormone assays. A subset of embryos was transferred to glass dishes and reared in clean water to evaluate developmental effects. The larvae (10 dpf) were randomly sampled, immediately frozen in liquid nitrogen, and stored at −80 °C for subsequent assaying of THs, protein expression, neurotransmitter, and acetylcholinesterase (AChE) activity. A subset of F1 larvae (10 dpf) was used for locomotor activity measurements. The hatching, malformation, survival, and growth rates were recorded for the F1 generation. Adult zebrafish were anesthetized in 0.03% MS-222 by prolonged immersion until cessation of opercular movement. Blood was collected from the caudal vein of each fish and transferred into heparin sodium-rinsed tubes. The liver, brain, and gonad tissues were excised, weighed and preserved at −80 °C until analysis. The study received the approval of the Institutional Animal Care and Use Committee and all animals were treated humanely with the aim of alleviating any suffering. They were maintained in accordance with guidelines for the care and use of laboratory animals of the National Institute for Food and Drug Control of China.

Y. Guo et al. / Science of the Total Environment 650 (2019) 557–565

2.3. Measurement of TH contents The blood samples from four fish of the same sex were pooled as a replicate (about 40 μL; n = 3), and plasma was stored at −80 °C until further analysis. The method for extraction of THs from eggs (200 eggs per replicate; n = 3) and larvae (200 larvae per replicate; n = 3) was implemented according to a previous procedure (Yu et al., 2011). Total TH levels (thyroxine, T4; 3,5,3′-triiodothyronine, T3) were measured using commercial ELISA kits (UscnLife Science Inc., Wuhan, China) following the manufacturer's instructions (Text S1 in Supplementary Materials). 2.4. Quantitative real-time PCR (qPCR) assay Four brains of adult fish of the same sex were pooled together as a replicate (n = 3). Approximately 100 offspring larvae at 10 dpf were collected to examine gene transcription (n = 3). Extraction, purification, and quantification of total RNA and first-strand cDNA synthesis were performed as described previously (Chen et al., 2017a). Primer sequences were designed using the Primer 3 software (http://frodo.wi. mit.edu/) and are listed in Table S1 in Supplementary Materials. Ribosomal protein L8 (Rpl8) was used as the reference gene to calculate the relative gene transcriptional levels based on the 2−ΔΔT method. 2.5. Protein extraction and western blot analysis Western blot analysis was carried out using approximately 200 larvae from each replicate (n = 3) according to a previously described method (Chen et al., 2012). Detailed procedures are provided in the Supplementary Material (Text S2). The expression levels of myelin basic protein (Mbp), α1-tubulin, and synapsin IIa (Syn2a) were selected for analysis. The rabbit Mbp antibody (AnaSpec, Fremont, CA), rabbit α1-tubulin antibody (Abcam, Cambridge, UK), and rabbit Syn2a antibody (Synaptic Systems, Göttingen, Germany) have previously been verified as reactive and suitable for zebrafish studies (Wang et al., 2015). Quantitation of protein expression was performed by densitometry, with the results normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. 2.6. Neurotransmitter levels in adult zebrafish brain and in larvae Extraction of neurotransmitters from adult fish brains (a pool of four brains of the same sex as a replicate; n = 3) and F1 larvae (50 larvae per replicate; n = 3) was performed as described previously (Tufi et al., 2016; Wang et al., 2012). Identification and quantification of analytes were performed using an Agilent 1200 (Agilent, USA) liquid chromatograph equipped with a triple quadrupole (Agilent, USA) tandem mass spectrometer. Dopamine, serotonin and acetylcholine were used as the analytical standards. A detailed description of the analytical procedure is provided in the Supplementary Material (Text S3).

559

following a previously described method (Chen et al., 2012). Larval swimming activity was monitored in a 24-well microplate (one larva per well) under a 15-min continuous light period and in response to light-dark-light transitions (5 min per photoperiod), respectively. A total of 30 larvae per treatment (10 larvae per replicate and three replicates per treatment) were recorded. Data (frequency of movements, distance traveled, and total duration of movements) were collected every 60 s, and further analyzed using custom Open Office.Org 2.4 software. 2.9. Quantification of BPA and n-TiO2 Chemical analysis of BPA and n-TiO2 in the exposure solutions, adult fish (one fish per replicate; n = 3) and F1 eggs (approximately 100 eggs per replicate; n = 3) were performed in accordance with previously published methods (Fang et al., 2016). Detailed protocols for the extraction, cleanup, analysis, and quality assurance and control (QA/QC) are provided in the Supplementary Materials (Text S4). 2.10. Statistical analyses All data are expressed as the mean ± standard error of mean (SEM) values of three replicates. The data were initially verified for normality and homogeneity of variance using the Kolmogorov-Smirnov and Levene's tests, respectively. Data will be log-transformed if necessary. Differences between the control and exposure groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's test using SPSS 13.0 software (SPSS; Chicago, IL). Differences between single exposure groups and co-exposure groups were compared using the Student's t-test. A P value of b0.05 was considered statistically significant. 3. Results 3.1. Toxicological endpoints in F1 generation Hatching rates were significantly delayed at 3 dpf in the F1 generation derived from parents exposed to 20 μg/L BPA both with (21.4%; P = 0.006) and without n-TiO2 (16.2%; P = 0.009), compared to solvent controls (Table 1). However, a notable increase in malformations (i.e., pericardial edema and axial spinal curvature) was observed at 5 dpf in F1 larvae after their parents were exposed to 20 μg/L BPA both with and without n-TiO2 (259%, P = 0.047 and 239%, P = 0.025, respectively), indicating that parental exposure to BPA alone or combined with n-TiO2 results in developmental toxicity in their offspring (Table 1). Additionally, the survival rate was significantly depressed compared to controls for 10 dpf progeny from parents exposed to 20 μg/L BPA with or without n-TiO2 (16.9%, P = 0.007 and 9.8%, P = 0.017, respectively; Table 1). A significant difference was also observed between the 10 dpf larvae derived from parents co-exposed to

2.7. AChE activity assay Adult fish brain samples (a pool of four brains of the same sex as a replicate; n = 3) and larvae (50 larvae per replicate; n = 3) were homogenized on ice in 60 vol (v/w) of tris-citrate buffer (50 mM Tris, 2 mM EDTA, and 2 mM EGTA [pH 7.4], adjusted with citric acid), and then centrifuged at 3000 ×g for 10 min at 4 °C. AChE enzyme activity was measured using a commercial kit (Nanjing Keygen Biotech, Co, Ltd., Nanjing, China) following the manufacturer's instructions. Protein concentrations were measured using the Bradford method. 2.8. Locomotor activity in larval offspring Quantification of larval locomotor activity was performed using a Video-Track system (ViewPoint Life Sciences, Montreal, Canada)

Table 1 Developmental parameters in the offspring after parental co-exposure to BPA and n-TiO2. Groups

Control 2 μg/L BPA 20 μg/L BPA 100 μg/L n-TiO2 2 μg/L BPA + n-TiO2 20 μg/L BPA + n-TiO2

Hatching (%)

Malformation Survival (%) (%)

Weight (mg)

3 dpf

5 dpf

10 dpf

10 dpf

82.3 ± 2.19 80.3 ± 2.31 69.0 ± 1.73* 82.7 ± 2.73 79.7 ± 2.03 64.7 ± 2.40*

1.67 ± 0.33 2.67 ± 0.67 4.00 ± 0.58* 2.33 ± 0.33 3.00 ± 0.58 4.33 ± 0.88*

88.7 ± 1.86 85.3 ± 2.03 80.0 ± 1.16* 82.3 ± 1.45 83.0 ± 1.53 73.7 ± 0.88*#

0.38 ± 0.01 0.37 ± 0.01 0.35 ± 0.01 0.36 ± 0.01 0.35 ± 0.01 0.33 ± 0.01*

All data are expressed as mean ± SEM of three replicates. *P b 0.05 indicates significant difference between exposure groups when compared to the solvent control, and # P b 0.05 indicates significant difference when comparing co-exposure groups the corresponding BPA group without n-TiO2.

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Table 2 Total T4 and T3 levels in F0 adult zebrafish and in F1 eggs and larvae after parental exposure to BPA and n-TiO2.a F0/F1

THs

Sex

Control

2 μg/L BPA

20 μg/L BPA

100 μg/L n-TiO2

2 μg/L BPA + n-TiO2

20 μg/L BPA + n-TiO2

F0

T4

Female Male Female Male

24.2 ± 1.27 22.0 ± 0.73 2.56 ± 0.03 2.22 ± 0.10 12.71 ± 0.16 0.96 ± 0.03 19.84 ± 0.88 2.17 ± 0.11

24.6 ± 1.48 21.4 ± 0.30 2.17 ± 0.11 2.03 ± 0.06 12.19 ± 0.93 0.96 ± 0.08 19.36 ± 1.62 1.92 ± 0.13

19.8 ± 0.49* 19.6 ± 1.05 2.15 ± 0.17 2.01 ± 0.08 9.32 ± 0.86* 0.93 ± 0.05 16.29 ± 0.56* 1.87 ± 0.09

23.5 ± 1.59 21.5 ± 0.64 2.41 ± 0.10 2.03 ± 0.12 11.96 ± 0.49 0.94 ± 0.02 17.62 ± 0.73 2.03 ± 0.08

22.3 ± 1.36 21.9 ± 0.70 2.15 ± 0.16 2.13 ± 0.17 10.63 ± 0.84 0.92 ± 0.11 16.61 ± 2.38 1.56 ± 0.20

15.9 ± 0.89*# 16.4 ± 1.20* 1.78 ± 0.10* 1.99 ± 0.12 6.34 ± 0.55*# 0.86 ± 0.11 12.33 ± 1.13*# 1.40 ± 0.23*

T3 Eggs F1 (10 dpf)

T4 T3 T4 T3

a TH levels are expressed as ng/mL in F0 zebrafish and as ng/g wet weight in F1 eggs and larvae, respectively. All data are expressed as means ± SEM of three replicates. *P b 0.05 indicates significant difference between exposure groups when compared to the solvent control, and # P b 0.05 indicates significant difference when comparing co-exposure groups to the corresponding BPA group without n-TiO2.

The expression levels of proteins essential for the development and differentiation of the central nervous system, including Mbp, Syn2a, and α1-tubulin, were examined in zebrafish larvae at 10 dpf after parental exposure. As shown in Fig. 1, the expression levels of the Mbp and Syn2a proteins, two biomarkers of axon myelination and synapse formation, respectively, were significantly lower in larvae derived from 20 μg/L BPA, n-TiO2, and the combined exposure groups, compared to the control groups. The expression of α1-tubulin, an intermediate filament protein associated with the cytoskeletal organization of developing neurons, was also significantly lower in larvae derived from the n-TiO2 and 20 μg/L BPA with n-TiO2 exposure groups (Fig. 1). Moreover, a marked reduction in Syn2a and α1-tubulin protein expression was also observed in larvae derived from the co-exposure groups compared to those derived from parents exposed to 20 μg/L BPA alone (Fig. 1).

2.0

Mbp

α1-tubulin

Syn2a

# &

1.5

#

1.0

*

* *

*

*

*

*

*

0.5

2

2

μ 20 g/L 0 2 μg B μg /L PA 20 /L B μg B P /L PA n-T A B + iO PA n 2 + TiO n- 2 Ti O

2

2

μ 20 g/L 0 2 μ μ g/ BP L A 20 g/L B μg B P P n /L A -T A B + iO PA n 2 + TiO n- 2 Ti O

0.0

2

The influence of BPA exposure with or without n-TiO2 on the levels of THs in F0 adult and F1 larvae are shown in Table 2. In females, the T4 concentration was significantly lower in the 20 μg/L BPA treatment group than the control group (P = 0.03, Table 2). Exposure to n-TiO2 alone did not induce alterations in T4 levels in comparison to the controls (Table 2). However, the T4 concentrations were significantly lower in the presence of n-TiO2 compared to the corresponding BPA (20 μg/L) alone (P = 0.017, Table 2). Compared to the control group, a significant decrease (30.5%, P = 0.002) in T3 levels was also observed in the group of females co-exposed to 20 μg/L BPA and n-TiO2. In males, we only observed significantly decreased T4 levels (25.5%, P = 0.016, Table 2) in the combined BPA (20 μg/L) and n-TiO2 group, when compared to the control group. In the F1 generation, T4 levels were significantly lower in eggs and larvae (10 dpf) derived from adults exposed to 20 μg/L BPA (26.7%, P = 0.018 and 17.9%, P = 0.027, respectively, Table 2). Moreover, parents co-exposed to n-TiO2 and 20 μg/L BPA resulted in further reductions in T4 content in the F1 progeny eggs and larvae compared to 20 μg/L BPA exposure alone (32.0%, P = 0.043 and 24.3%, P = 0.035, respectively; Table 2). In terms of T3 levels, no significant differences were observed in the eggs or larvae derived from parents exposed to BPA, n-TiO2 or combination exposure, with the exception of the coexposure group of 20 μg/L BPA with n-TiO2 for 10 dpf larvae (Table 2). In the brains of adult parents (Fig. S1A, Supplementary Materials), significant upregulation of the transcription of the thyroid-stimulating hormone subunit beta (TSHβ) and corticotropin-releasing hormone (CRH) genes indicated the initiation of a positive feedback loop along the thyroidal axis to increase the synthesis of THs, which adaptively compensated for the decreased TH levels in plasma. In the offspring larvae, positive regulation of the feedback mechanism was also observed, as manifested by the upregulation of TSHβ and CRH gene transcription in the 20 μg/L BPA alone group and/or the co-exposure group treated with 20 μg/L BPA and n-TiO2 in comparison with the control (Fig. S1B). Gene transcription of 5′-deiodinase 2 (DIO2), which plays pivotal roles in the conversion of the T4 prohormone to bioactive T3, was also significantly increased in larval offspring by parental exposure to 20 μg/L BPA alone and the 20 μg/L BPA and n-TiO2 combination relative to the control group (Fig. S1B). Significant differences were found in the TSHβ, CRH and DIO2 values when the 20 μg/L BPA group was compared with the combined 20 μg/L BPA and n-TiO2 group in larval offspring. However, genes associated with TH synthesis, including thyroglobulin (TG), thyroid peroxidase (TPO) and sodium-iodide symporter (NIS), were consistently down-regulated in offspring larvae

3.3. Protein expression in F1 larvae

2 μ 20 g/ 2 μg L B 0 μg /L PA 20 /L B μg B P /L PA n-T A B + iO PA n 2 + TiO n- 2 Ti O

3.2. TH levels and thyroidal gene transcription in adults and offspring

derived from the 20 μg/L BPA alone group and/or the co-exposure group of 20 μg/L BPA with n-TiO2 relative to the control group (Fig. S1C).

Optical density

20 μg/L BPA and n-TiO2 compared to 20 μg/L BPA alone (7.88%, P = 0.012; Table 1). Moreover, the growth of larvae at 10 dpf from parents co-exposed to n-TiO2 and 20 μg/L BPA was inhibited (13.2%, P = 0.029) relative to the control specimens (Table 1).

Fig. 1. Western blot analysis demonstrating decreases in expression levels of critical neural proteins (Mbp, Syn2a, and α1-tubulin) in 10-dpf F1 larvae derived from exposed parents to different combinations of n-TiO2 and BPA (0, 2, and 20 μg/L). The data represent the mean ± SEM of three replicate samples. *P b 0.05 indicates significant differences between exposure groups and the corresponding control group, while # P b 0.05 indicates a significant difference when comparing co-exposure groups to the corresponding BPA groups without n-TiO2, and & P b 0.05 indicates significant differences between coexposure groups and the n-TiO2 group.

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561

3.4. Neurotransmitter levels and AChE activity

(A) 600

Neurotransmitter (ng/g ww)

serotonin

dopamine

400

acetylcholine

&

* * *

*

200

2

2

2

2 μ 20 g/L 0 2 μg B μg /L PA 20 /L B μg B P /L PA n-T A B + iO PA n 2 + TiO n- 2 Ti O

2 μ 20 g/L 0 2 μg B μg /L PA 20 /L B μg B P P /L A n-T A B + iO PA n 2 + TiO n- 2 Ti O

0 2 μ 20 g/ 2 μg L B 0 μg /L PA 20 /L B μg B P P /L A n-T A B + iO PA n 2 + TiO n- 2 Ti O

We further examined the dopamine, serotonin, and acetylcholine levels in adult zebrafish brains and larvae. In the brains of the female adults, we observed significant reductions in serotonin (19.3% and 30.4%; P b 0.05) and dopamine (27.1% and 28.2%; P b 0.05) in the nTiO2 alone group and the 20 μg/L BPA co-exposure group relative to the controls, while no significant differences were observed in the acetylcholine content between the treatment groups (Fig. 2A). In adult male brains, long-term exposure did not alter the content of any of the neurotransmitters tested in any of the treatment groups (see Supplementary Materials, Fig. S2). Larvae derived from fish exposed to nTiO2, 20 μg/L BPA, or both exhibited significantly decreased amounts of serotonin (20.9%, 21.5%, and 29.3%; P b 0.05) and dopamine (30.8%, 28.8%, and 37.4%; P b 0.05) compared to controls (Fig. 2B). Larval levels of acetylcholine were significantly lower after pre-parental co-exposure to 20 μg/L BPA and n-TiO2 when compared with controls or the corresponding 20 μg/L BPA exposure group (29.6%, P = 0.01; 17.2%, P = 0.001, respectively; Fig. 2B). No significant difference in AChE activity was observed in adult male and female brains between any treatment groups (see Supplementary Materials, Fig. S3). However, AChE activity was significantly lower in larvae derived from co-exposed parents to 20 μg/L BPA and n-TiO2 compared to control and the corresponding 20 μg/L BPA alone groups (17.5%, P = 0.019; 22.2%, P = 0.029, respectively; Fig. 2C).

(B) 3.5. Locomotor behavior of zebrafish larvae

Neurotransmitter (ng/g ww)

serotonin

dopamine

#

* *

40

*

20

2

2

2

2 μ 20 g/L 0 2 μg B μ /L P A 20 g/L B μg B P P /L A n- A B + TiO PA n - 2 + TiO n- 2 Ti O

(C) 1.5

#

1.0

* 0.5

/L

nTi O

2

B PA

2

B PA

+

μg

μ

g/ L

20 20

2

μ

g/ L

2

B PA

+

μg

/L

nTi O

2

B PA

0.0 nTi O

Fig. 2. Neurotransmitter contents (dopamine, serotonin, and acetylcholine) were decreased in female brains exposed to BPA (0, 2, and 20 μg/L) alone or in combination with n-TiO2 for four months (A), as well as in 10-dpf F1 larvae from exposed parents (B). The levels of neurotransmitters are expressed as ng/g ww (ww = wet weight). All data are expressed as mean ± SEM of three replicate samples. *P b 0.05 indicates significant differences between exposure groups and the corresponding control group, while # P b 0.05 indicates a significant difference when comparing co-exposure groups to the corresponding BPA groups without n-TiO2, and & P b 0.05 indicates significant differences between co-exposure groups and the n-TiO2 group.

2 μ 20 g/L 0 2 μg B μg /L P A 20 /L B μg B P P /L A n- A B + TiO PA n - 2 + TiO n- 2 Ti O

0

0

The actual concentrations of BPA and n-TiO2 were measured just after (T0) and before (T24) renewal of the exposure solutions. After renewal, the measured BPA and n-TiO2 concentrations were found to closely match the desired nominal concentrations (Fig. S4). However, after 24 h of exposure, the measured BPA and n-TiO2 concentrations had significantly decreased (Fig. S4A for n-TiO2, and Fig. S4B for BPA). In the groups that were co-exposed to BPA and n-TiO2, the presence of BPA did not induce significant changes in the waterborne concentrations of n-TiO2 particles relative to the n-TiO2 alone group (Fig. S4A). However, the BPA concentration decreased further after addition of nanoparticles compared to the corresponding concentrations in the BPA alone group (Fig. S4B). Concentrations detected in the control subjects were below the LOD.

*

* * *

AChE activity (μmol/min/mg protein)

3.6. Quantification of BPA and n-TiO2

acetylcholine

60

2 μ 20 g/ 2 μg L B 0 μg /L P A 20 /L B μg B P /L PA n- A B + TiO PA n - 2 + TiO n- 2 Ti O

In continuous light conditions, swimming speed was significantly reduced in 10 dpf larvae derived from n-TiO2 alone, 20 μg/L BPA alone, and the co-exposure groups when compared with the control group (21.6%, 16.1%, and 30.8%; P b 0.05, Fig. 3A). Moreover, a significant difference in speed was also observed between the co-exposure 20 μg/L BPA and n-TiO2 group versus the 20 μg/L BPA alone group (17.5%, P = 0.036; Fig. 3A). During light-dark transition stimulation, the average swimming speed was significantly lower during both the light and dark periods in 10 dpf larvae derived from n-TiO2 alone, 20 μg/L BPA alone, and the co-exposure group, when compared with the control equivalents (Fig. 3B). During the light periods, a notable further decrease in swimming speed was observed in the co-exposure to 20 μg/L BPA/n-TiO2 group relative to the 20 μg/L BPA alone group (Fig. 3B).

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(A)

0.20

*

*

Female

400

Content of TiO2 (μg/g d.w.)

Average speed (mm/s)

(A)

#

0.25

*

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*

*

*

300

Eggs

*

*

*

200

100

2

2

2

2

20

+

μg /L 20

2

μg 20 /L μg BP n 0 /L A -Ti B + n O2 PA T + iO n- 2 Ti O 2 2 μg 20 /L μg BP n 0 /L A -Ti B + n O2 PA -T + iO n- 2 Ti O

μg /L μg BP n 0 /L A -Ti B + n O2 PA -T + iO n- 2 Ti O

nTi O

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B PA

0

B PA

20

+ B PA

μg /L

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μg /L

nTi O

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nTi O

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800

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*

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* *

*

*

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Average speed (mm/s)

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# #

400

200

# #

#

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2 μ 20 g/L 0 2 μg B μg /L PA 20 /L B μg B P /L PA n- A B +n TiO PA T 2 +n iO -T 2 iO

The levels of n-TiO2 detected in F0 adults and F1 eggs are shown in Fig. 4A. In the controls and after BPA exposure alone, the concentration of Ti was below the LOD. Obvious accumulation of Ti was observed in both males and females after long-term exposure to 100 μg/L n-TiO2 (Fig. 4A). Moreover, BPA significantly altered the bioavailability of Ti in fish, as indicated by the significantly increased body burden in the co-exposure groups (Fig. 4A). The content of Ti in F1 eggs was similar to the maternal BPA and n-TiO2 co-exposure group (Fig. 4A). The total body burden of BPA in F0 adult males and females showed a concentration-dependent relationship in the 2 and 20 μg/L exposure groups (Fig. 4B). Likewise, a concentration-dependent increase of BPA was also observed in the co-exposure groups in both sexes (Fig. 4B). Compared with BPA alone, significant increases were observed in the co-

2

μg /L 2 B μg 20 P /L μg 2 A + B 0 /L 0 n- PA B μg TiO PA /L + BP 2 n- A Ti 2 O μg 2 /L 2 B μg 20 PA /L 0 μg 2 + B /L 0 n- PA B μg TiO PA /L + BP 2 n- A Ti 2 O μg 2 /L 2 μ B g 20 P /L μg 2 A + B 0 /L 0 n- PA B μg TiO PA /L + BP 2 n- A Ti O

Fig. 3. Lethargic locomotor behavior in F1 larvae derived from exposed parents to BPA (0, 2, and 20 μg/L) alone or in combination with n-TiO2 for four months. Average swimming speed during a continuous light test (A), and average swimming speed of the larvae during a light-dark-light photoperiod stimulation test (B) are presented. Data are expressed as the mean ± SEM of three replicates (10 larvae per replicate). *P b 0.05 indicates significant differences between exposure groups and control group, and # P b 0.05 indicates significant difference when comparing co-exposure groups to the corresponding BPA groups without n-TiO2, and & P b 0.05 indicates significant differences between co-exposure groups and the n-TiO2 group.

0

2

2

2 μ 20 g/L 0 2 μg B μg /L PA 20 /L BP μg B P /L A n- A B +n TiO PA T 2 +n iO -T 2 iO

2

Male

#

0.0 2 μ 20 g/ 2 μg L B 0 μg /L PA 20 /L B μg B P /L PA n- A B +n TiO PA T 2 +n iO -T 2 iO

Female

Fig. 4. Reciprocal facilitation of n-TiO2 (A) and BPA (B) accumulation in female and male zebrafish after co-exposure as well as enhanced transfer of pollutants in the F1 eggs from co-exposed parents. Data are expressed as the mean ± SEM of three replicate samples. The measured Ti and BPA contents for the control were below the LOD. For Ti, a significant difference between co-exposure groups and n-TiO2 alone is indicated by *P b 0.05. For BPA, # P b 0.05 indicates significant difference when comparing co-exposure groups to the corresponding BPA groups without n-TiO2. dw = dry weight.

exposure groups (Fig. 4B). Furthermore, we observed concentrationdependent increases in BPA levels in F1 eggs derived from parental exposure to BPA in the 2 and 20 μg/L BPA alone groups, while the accumulation of BPA further increased in F1 eggs derived from parents co-exposed to nTiO2 and BPA at both concentrations (Fig. 4B). These results further demonstrated that n-TiO2 and BPA can bioaccumulate in adult females and males, and have the ability to transfer to their offspring. 4. Discussion In the present study, we demonstrated that BPA and n-TiO2 were bioconcentrated in exposed adult zebrafish, and that the compounds

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were then transferred to their offspring following parental exposure. The presence of n-TiO2 can significantly increase the body burden of BPA, leading to enhanced adverse effects on the thyroid endocrine system in both generations. The development of the offspring nervous system was also impaired after parental exposure. A previous study demonstrated that BPA and n-TiO2 can be detected in adult gonad tissue after co-exposure (Fang et al., 2016). Consistently, the present study also observed high body burden of BPA and n-TiO2 in adult fish, confirming the bioconcentration and bioavailability of these chemicals. Additionally, significant levels of BPA and n-TiO2 were transferred to eggs after parental exposure. In co-exposed zebrafish, we observed further increases in n-TiO2 and BPA content relative to their corresponding single exposure group, suggesting that n-TiO2 can act as a carrier of BPA and, in turn, BPA can affect the uptake of n-TiO2 (Fang et al., 2016). It should be noted that a higher burden tendency of BPA and n-TiO2 was observed in females than in males, which may originate from gender differences in uptake, distribution, metabolism, and elimination of pollutants due to the inherent distinction in hormonal levels and detoxifying capacity (Chen et al., 2018b; Marques et al., 2013). Decreased plasma T4 or T3 levels were observed in adult zebrafish after exposure to 20 μg/L BPA with or without 100 μg/L n-TiO2, indicating the thyroid endocrine disrupting effects of BPA in fish. Studies on zebrafish embryo-larvae show that BPA significantly upregulates the transcription of genes involved in TH synthesis and the feedback loop along the HPT axis (Chan and Chan, 2012; Gentilcore et al., 2013; Lu et al., 2018), resulting in thyroid-disrupting effects. In the present study, the increased transcription of the CRH and TSHβ genes implies that the positive feedback regulation occurs as a response to the reduced plasma levels of THs, which can adaptively increase the synthesis of THs. Furthermore, the increased reduction of TH levels and the increased upregulation of the TSHβ gene after co-exposure to n-TiO2 and 20 μg/L BPA may be attributed to the increased body burden of BPA caused by n-TiO2. The present study also demonstrated sexdependent alterations of THs in adult zebrafish after co-exposure to nTiO2 and BPA. Similar sex-specific effects are also reported for teleosts after chronic exposure to other pollutants (Chen et al., 2018b; Wang et al., 2015; Yu et al., 2011), highlighting the need to incorporate sexspecificity in environmental risk assessment. Fish eggs contain a substantial concentration of THs of maternal origin that are essential to early embryogenesis (Campinho et al., 2014). This study demonstrated that parental exposure to 20 μg/L BPA with or without 100 μg/L n-TiO2 resulted in significantly reduced T4 levels in eggs, which could be due to the lower maternal plasma levels of T4. Lower levels of T4 will be transferred to eggs from maternal zebrafish with hypothyroidism (Wang et al., 2015). Although there is a significant increase in T4 production from 72 h post fertilization (hpf) to counteract the diminished pool of maternal T4 (Porazzi et al., 2009), we still observed a reduction of THs in 10-dpf F1 larvae derived from exposed parents, suggesting a state of hypothyroidism in unexposed offspring following parental exposure. Despite the activation of positive feedback regulation (TSHβ and CRH), the inability of F1 larvae to reverse parental influences may indicate significantly compromised fitness as a result of exposure to a BPA and n-TiO2 mixture (Chen et al., 2017a). A high concentration of BPA in F1 eggs may affect the synthesis of THs during embryonic development; this is supported by current results about the downregulation of TH-synthetic genes (TG, TPO, NIS and DIO2). THs play pivotal roles in vertebrate development and physiology, especially in the developing brain, which is believed to be extremely sensitive to many xenobiotics (Chan and Chan, 2012). Furthermore, it has been demonstrated that n-TiO2 can be translocated to the zebrafish brain and lead to brain injury, thus causing neurotoxicity even at low doses (Sheng et al., 2016). Reciprocal enhancement of BPA and n-TiO2 bioaccumulation in zebrafish brains has also been noted after coexposure (Fang et al., 2016). Therefore, both the reduced TH levels along with the transfer of BPA and n-TiO2 to eggs and larvae may impair the development of the central nervous system (CNS), which could

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consequently lead to adverse impacts on the behavior and health of the progeny. In the present study, developmental neurotoxicity in the offspring was caused by parental single or mixed exposure to BPA and n-TiO2, as indicated by reduced behavioral activity. Locomotor behavior is considered as a sensitive indicator of neurotoxicity in developing zebrafish (Drapeau et al., 2002). The presented observations are in agreement with the altered behavioral performance previously reported in BPA- or n-TiO2-exposed zebrafish larvae (Kinch et al., 2015; Saili et al., 2012). Furthermore, it is intriguing that the presence of 2 μg/L BPA diminished the neurotoxic effects of n-TiO2, while coexistence of BPA at a high concentration (20 μg/L) with n-TiO2 led to enhanced neurotoxicity. These dual modes of interaction indicate the importance of exposure concentrations when assessing the interaction between organic pollutants and suspended particles. Similar findings have also been reported in previous co-exposure studies, which speculate that the varied interactive modes are caused by the specific adsorptive effect of organic pollutants on suspended particles at different exposure concentrations, thus affecting the disassociation dynamics from the complexes and subsequently interfering with their bioavailability and toxicity (Chen et al., 2018a; Chen et al., 2017a, 2017b). To explore the potential mechanism underpinning behavioral lethargy, we analyzed the levels of serotonin and dopamine monoamine neurotransmitters in the adult zebrafish and larval offspring. Serotonin is known to participate in motor control, cognition, and motivation, while dopamine can promote the specification, differentiation, and phenotype maintenance of hippocampal neurons (Fricker et al., 2005; Iversen and Iversen, 2007). The present study observed significant decreases in serotonin and dopamine in the brains of female parents after single exposure to n-TiO2 and co-exposure with 20 μg/L BPA. A previous study also demonstrated that exposure to increased concentrations of nTiO2 results in significant reductions in serotonin and dopamine in zebrafish brain (Sheng et al., 2016). Furthermore, parental single or combined exposure to n-TiO2 and BPA significantly decreased basal levels of serotonin and dopamine in F1 zebrafish larvae. The reductions in the levels of serotonin and dopamine may impact normal functioning of the neurotransmitter system, ultimately affecting neurobehavior in zebrafish larvae. Additionally, acetylcholine levels and AChE activity were reduced in F1 larvae derived from co-exposed parents. As a major neurotransmitter in the cholinergic system, acetylcholine plays a critical role in modulating motor and cognitive functions (Driscoll et al., 2009), and is maintained by the hydrolysis of AChE in zebrafish (Behra et al., 2002). The decreased AChE activity and lower levels of acetylcholine may interfere with neural signaling, leading to abnormal muscular contraction and swimming behavior (Chen et al., 2017a, 2017b). Another important factor that may lead to developmental neurotoxicity is modified expression of proteins that are crucial in the CNS. Mbp protein is a biomarker of axon myelination in the developing CNS of zebrafish (Brosamle and Halpern, 2002), and α1-tubulin is a neuronspecific microtubule whose expression is induced in the developing and regenerating CNS (Fausett and Goldman, 2006). Accordingly, down-regulated levels of Mbp and α1-tubulin proteins observed in the present study may have adverse effects on brain architecture and function. Additionally, Syn2a is a neuronal phosphoprotein that binds small synaptic vesicles to induce further synaptogenesis (Kao et al., 1998). Recently, it was demonstrated that Syn2a is widely expressed in the CNS of zebrafish during nervous system differentiation, indicating that synapsins play important roles during early developmental stages, such as neuronal differentiation and synaptogenesis (Garbarino et al., 2014). Therefore, the down-regulation of Syn2a observed herein may affect synaptogenesis and neuronal differentiation, and eventually disrupt neuronal functions. Similar results have also been reported for zebrafish and rats after chemical exposure (Ramarao et al., 2011; Wang et al., 2015). The present study demonstrated that parental co-exposure to BPA and n-TiO2 could reciprocally enhance their bioaccumulation in adults and facilitate the transfer of pollutants to their progeny. Environmental co-existence of BPA with n-TiO2 enhanced the thyroid-disrupting

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effects and developmental neurotoxicity in larval offspring. In addition to BPA (Yan et al., 2014; Fang et al., 2016), TiO2 nanoparticles are frequently found to adsorb various organic pollutants and subsequently vary their innate toxicological effects, including pentachlorophenol (Fang et al., 2015), phenanthrene (Tian et al., 2014), BDE-209 (Wang et al., 2014), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; Canesi et al., 2014), and tributyltin (Zhu et al., 2011). The dynamic interaction between nanoparticles and organic pollutants underlines that a comprehensive risk assessment should not only consider the direct effects of pollutants, but also integrate their indirect interaction. In a co-exposure condition that includes nanoparticles and organic pollutants, the enhanced transfer of toxins from exposed parents to their offspring is supposed to significantly complicate the workflow of environmental risk assessment. Acknowledgements This work was supported by the National Natural Science Foundation of China [grant number 21577168], the Strategic Priority Research Program of the Chinese Academy of Sciences [grant number XDB14040103], and the State Key Laboratory of Freshwater Ecology and Biotechnology (2016FBZ11). Conflicts of interest The authors have no conflicts of interest to declare. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.09.007. References Behra, M., Cousin, X., Bertrand, C., Vonesch, J.L., Biellmann, D., Chatonnet, A., et al., 2002. Acetylcholinesterase is required for neuronal and muscular development in the zebrafish embryo. Nat. Neurosci. 5, 111–118. Bhandari, R.K., Deem, S.L., Holliday, D.K., Jandegian, C.M., Kassotis, C.D., Nagel, S.C., Tillitt, D.E., vom Saal, F.S., Rosenfeld, C.S., 2015. Effects of the environmental estrogenic contaminants bisphenol A and 17 alpha-ethinyl estradiol on sexual development and adult behaviors in aquatic wildlife species. Gen. Comp. Endocrinol. 214, 195–219. Brosamle, C., Halpern, M.E., 2002. Characterization of myelination in the developing zebrafish. Glia 39, 47–57. Campinho, M.A., Saraiva, J., Florindo, C., Power, D.M., 2014. Maternal thyroid hormones are essential for neural development in zebrafish. Mol. Endocrinol. 28, 1136–1149. Canesi, L., Frenzilli, G., Balbi, T., Bernadeschi, M., Ciacci, C., Corsolini, S., Della Torre, C., Fabbri, R., Faleri, C., Focardi, S., Guidi, P., 2014. Interactive effects of n-TiO2 and 2,3,7,8-TCDD on the marine bivalve Mytilus galloprovincialis. Aquat. Toxicol. 153, 53–65. Chan, W.K., Chan, K.M., 2012. Disruption of the hypothalamic-pituitary-thyroid axis in zebrafish embryo-larvae following waterborne exposure to BDE-47, TBBPA and BPA. Aquat. Toxicol. 108, 106–111. Chen, L., Yu, K., Huang, C., Yu, L., Zhu, B., Lam, P.K., et al., 2012. Prenatal transfer of polybrominated diphenyl ethers (PBDEs) results in developmental neurotoxicity in zebrafish larvae. Environ. Sci. Technol. 46, 9727–9734. Chen, L., Wang, X.F., Zhang, X.H., Lam, P.K.S., Guo, Y.Y., Lam, J.C.W., Zhou, B.S., 2017a. Transgenerational endocrine disruption and neurotoxicity in zebrafish larvae after parental exposure to binary mixtures of decabromodiphenyl ether (BDE-209) and lead. Environ. Pollut. 230, 96–106. Chen, Q., Gundlach, M., Yang, S., Jiang, J., Velki, M., Yin, D., Hollert, H., 2017b. Quantitative investigation of the mechanisms of microplastics and nanoplastics toward zebrafish larvae locomotor activity. Sci. Total Environ. 584-585, 1022–1031. Chen, L., Guo, Y., Hu, C., Lam, P.K.S., Lam, J.C.W., Zhou, B., 2018a. Dysbiosis of gut microbiota by chronic coexposure to titanium dioxide nanoparticles and bisphenol A: implications for host health in zebrafish. Environ. Pollut. 234, 307–317. Chen, L., Hu, C., Tsui, M.M.P., Wan, T., Peterson, D.R., Shi, Q., Lam, P.K.S., Au, D.W.T., Lam, J.C.W., Zhou, B., 2018b. Multigenerational disruption of the thyroid endocrine system in marine medaka after a life-cycle exposure to perfluorobutanesulfonate. Environ. Sci. Technol. 52, 4432–4439. Chong, M.N., Jin, B., Chow, C.W., Saint, C., 2010. Recent developments in photocatalytic water treatment technology: a review. Water Res. 44, 2997–3027. Ding, K.K., Kong, X.T., Wang, J.P., Lu, L.P., Zhou, W.F., Zhan, T.J., Zhang, C.L., Zhuang, S.L., 2017. Side chains of parabens modulate antiandrogenic activity: in vitro and molecular docking studies. Environ. Sci. Technol. 51, 6452–6460. Drapeau, P., Saint-Amant, L., Buss, R.R., Chong, M., McDearmid, J.R., Brustein, E., 2002. Development of the locomotor network in zebrafish. Prog. Neurobiol. 68, 85–111.

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