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Effects of perinatal exposure to BPA, BPF and BPAF on liver function in male mouse offspring involving in oxidative damage and metabolic disorder
Accepted Manuscript Effects of perinatal exposure to BPA, BPF and BPAF on liver function in male mouse offspring involving in oxidative damage and metabolic disorder Zhiyuan Meng, Sinuo Tian, Jin Yan, Ming Jia, Sen Yan, Ruisheng Li, Renke Zhang, Wentao Zhu, Zhiqiang Zhou PII:
S0269-7491(18)35272-2
DOI:
https://doi.org/10.1016/j.envpol.2019.01.116
Reference:
ENPO 12155
To appear in:
Environmental Pollution
Received Date: 22 November 2018 Revised Date:
29 January 2019
Accepted Date: 29 January 2019
Please cite this article as: Meng, Z., Tian, S., Yan, J., Jia, M., Yan, S., Li, R., Zhang, R., Zhu, W., Zhou, Z., Effects of perinatal exposure to BPA, BPF and BPAF on liver function in male mouse offspring involving in oxidative damage and metabolic disorder, Environmental Pollution (2019), doi: https:// doi.org/10.1016/j.envpol.2019.01.116. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effects of perinatal exposure to BPA, BPF and BPAF on liver
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function in male mouse offspring involving in oxidative damage and
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metabolic disorder
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Zhiyuan Meng a, Sinuo Tian a, Jin Yan a, Ming Jia a, Sen Yan a, Ruisheng Li a, Renke
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Zhang a, Wentao Zhu a *, Zhiqiang Zhou a
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a
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Department of Applied Chemistry, China Agricultural University, Beijing 100193,
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China
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Beijing Advanced Innovation Center for Food Nutrition and Human Health,
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* Corresponding author: Yuanmingyuan west road 2, Beijing 100193, PR China.
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E-mail address: [email protected]. (W. Zhu)
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ACCEPTED MANUSCRIPT Abstract: Bisphenols (BPs) are common environmental pollutants that are ubiquitous
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in the natural environment and can affect human health. In this study, we explored the
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effects of perinatal exposure to BPA, BPF and BPAF on liver function involving in
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oxidative damage and metabolic disorders in male mouse offspring. We found that
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BPA exposure impairs the antioxidant defense system, increases lipid peroxidation,
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and causes oxidative damage in the liver. Furthermore, the levels of 13 metabolites
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were significantly altered following BPA exposure. We found that BPF exposure
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significantly increased the expression and activity of CAT, suggesting disturbances in
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the antioxidant defense system. Moreover, BPF exposure led to metabolic disorders in
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the liver due to changes in the levels of 8 key metabolites. Exposure to BPAF caused
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no negative effects on oxidative damage, but altered the levels of β-glucose and
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glycogen. In summary, perinatal exposure to BPA, BPF and BPAF differentially
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influence oxidative damage and metabolic disorders in the livers of male mouse
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offspring. The impact of early life exposure to BPs now warrants future
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investigations.
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Capsule: Perinatal exposure to BPA, BPF and BPAF differentially influence oxidative
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damage, metabolic disorders, and subsequent liver function in male mouse offspring.
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Keywords: bisphenol A; bisphenol F; bisphenol AF; oxidative damage; metabolic
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disorder.
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1. Introduction Bisphenols (BPs) are important chemical substances that are widely used in the
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synthesis of plastics, particularly polycarbonates and epoxy resins. As the most
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commonly used bisphenol compound, bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)
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propane) is used in the production of plastic bottles, food plastic bags, medical
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materials, thermal paper and other products (Geens et al., 2012; Michalowicz, 2014).
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In 2015, the value of BPA used for the production of plastic monomers and
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plasticizers exceeded six billion pounds, making it one of the highest-volume
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chemicals worldwide. Due to its use in industrial applications, BPA enters the
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environment through various channels, leading to environmental risks. BPA has been
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reported to be distributed in water, soil, sediment, fish and foodstuffs, with the content
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of BPA reported as 0.08-0.47 µg/L in the surface waters of North America and Europe
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(Klecka et al., 2009). In China, the content of BPA is 0.04-0.64 µg/L in the Pearl
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River water and 0.88 µg/L in Guangzhou city water (Zhang and Zeng, 2016). The
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levels of BPA in fish liver and muscle tissue were also reported as 2-75 µg/kg and
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1-11 g/kg, respectively (Mita et al., 2011). In recent years due to increased toxicology
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and population epidemiology studies, the health risks of BPA have attracted
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widespread attention from both regulators and consumers. In the United States, the
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European Union, and other countries, the use of BPA is prohibited in food packaging
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products such as bottles (Michalowicz, 2014). Alternatives to BPA including
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bisphenol F (BPF, bis (4-hydroxyphenyl) methane) and bisphenol AF (BPAF,
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4,4’-(Hexafluoroisopropylidene) diphenol) have been increasingly used in industrial
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applications. Subsequently, BPF and BPAF have been detected in vegetables, meat,
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and seafood (Chen et al., 2016; Liao and Kannan, 2011, 2013). Importantly, humans can contact and absorb BPs from the natural environment.
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The average concentration of BPA detected in the urine samples of Belgian adults was
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3.9 ng/mL (Pirard et al., 2012). BPA was also detected in the urine of pregnant women
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during the first and second trimester (1.39 ng / mL and 1.27 ng / mL, respectively)
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(Chiu et al., 2017). Similarly to BPA, BPF and BPAF have been detected in the blood
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and adipose tissue of humans (Calafat et al., 2009; Mokra et al., 2018; Yang et al.,
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2012). The ability of BPA to disrupt the endocrine system has also been reported in
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numerous studies (Vandenberg et al., 2016). Similarly, adverse effects on the male
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reproductive system (Karnam et al., 2014) and thyroid function (Wang et al., 2015)
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have been documented. In addition, long-term exposure to BPA has been suggested to
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induce hypertension, diabetes, insulin resistance, obesity and heart disease (Cabaton
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et al., 2011; Sengupta et al., 2013; Shankar et al., 2012). Recent studies document that
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BPF and BPAF lead to adverse health effects and at low concentrations and can
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induce DNA damage (Mokra et al., 2018). Perinatal exposure to BPAF has also been
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shown to disrupt glycolipid homeostasis in the liver of female mouse offspring (Meng
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et al., 2018).
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Oxidative damage is a specific manifestation of environmental pollution that
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causes harm to humans and other organisms. The damage occurs as a result of
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excessive reactive oxygen species (ROS) due to an imbalance of oxidizing substances
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and the antioxidant system (Hirooka et al., 2010). Studies have shown that BPA
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Elobeid, 2012). Moreover, exposure to BPF causes oxidative stress in the immune
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system of juvenile common carp (Cyprinus carpio) (Qiu et al., 2018). In vitro studies
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have shown that BPAF has a negative impact on oocyte maturation in mice through
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oxidative stress and DNA damage (Ding et al., 2017). Studies on the association of
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oxidative damage and metabolic disorders induced by BPA, BPF and BPAF exposure
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have not been reported.
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In this study, we assessed the effects of BPA, BPF and BPAF on the liver
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function of male mouse offspring, including oxidative damage and metabolic
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disorders, following perinatal exposure. Specifically, we examined changes in the
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activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase
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(GSH-Px), changes in the levels of glutathione (GSH) and malondialdehyde (MDA),
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and alterations in the relative expression of oxidative stress-related genes including
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Sod1, Sod2, Cat, Gpx1, Gpx2, Nrf2, HO-1, and GADD45β in the liver tissue of male
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mouse offspring. In addition, the liver metabolic profiles of the offspring were
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assessed using 1H NMR. The results of this study provide a theoretical basis for
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further comprehensive analysis of the health risks that result from perinatal exposure
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to BPs.
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2. Materials and Methods
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2.1. Animals and treatments
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A total of 32 primigravida pregnant ICR mice were obtained from the Peking
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University Health Science Center (Beijing, China). Mice were maintained in
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and a normal diet (Keao Xieli Feed Co., Ltd. Beijing, China). Based on the mother's
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weight, BPA, BPF, and BPAF corn oil solutions were orally administered to pregnant
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mice (8 pregnant mice per group) at a dose of 100 ng/g bw/day from the 7th day of
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pregnancy to the 21st day after delivery. Control mice received only corn oil treatment.
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Each dam was culled to six male cubs the day after birth. Male offspring in each
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treatment group were provided a normal diet for 10 weeks after weaning at day 21.
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The specific experimental design is shown in Fig.1. Body weights were measured
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once a week and liver tissue and serum samples were collected at the end of this study.
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Samples were stored at −80℃ for further analysis.
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2.2. Analysis of biochemical parameters
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Serum samples from mice offspring at 13 weeks were collected for the detection
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of ALT and AST activity using commercial assay kits. The contents of triglyceride
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(TG) and total cholesterol (T-Cho) in the liver samples was also quantified. For the
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assessment of oxidative parameters, liver tissue (100 mg) was homogenized in 900 µL
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of saline and precipitates were collected by centrifugation at 3000 rpm for 12 min.
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Supernatants were collected for the assessment of SOD, CAT and GSH-Px activity
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and the levels of MDA and GSH using commercial analysis kits (Nanjing Jiancheng
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Bioengineering Institute, Nanjing, China) according to the manufacturer’s
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instructions.
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2.3. Histopathological analysis
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Liver sections from 13 week old mice were embedded in paraffin for histological
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examination. Sections of 5-6 µm were stained with hematoxylin and eosin (H & E)
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and analyzed on an Olympus microscope.
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2.4. Analysis of mRNA expression Liver samples were frozen in liquid nitrogen and grounded. TRIzol reagent
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(Tiangen Biotech Co., LTD., Beijing, China) was used to extract total RNA from the
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liver samples. cDNA was synthesized from total RNA (1.5 µg) using Fast Quant RT
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Kit (Tiangen Biotech Co., LTD., Beijing, China). qRT-PCR was performed using
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SuperReal PreMix Plus (SYBR Green) (Qiagen, China) and the Bio-Rad CFX 96
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PCR system (Bio-Rad, USA), respectively. Gene expression was normalized to
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β-actin. Primers were obtained from Sangon Biotech (Shanghai, China) and are listed
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in Table S1.
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2.5. Liver metabolomics analysis
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The preparation of liver samples for 1H NRM analysis was performed as
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previously described (Meng et al., 2018) and detailed in supplementary information.
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Billerica, MA, USA). Multivariate statistical analysis was performed using SIMCA P
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(Version 11, Umetrics, Sweden). 1H NMR parameters and multivariate statistical
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analysis were performed as previously described and outlined in Supporting
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information S2 (Zhang et al., 2015; Wang et al., 2018).
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2.6. Statistical analysis
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H NMR analysis were performed on a Bruker AV III600 NMR spectrometer (Bruker,
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All values are expressed as the mean ± SD. Statistical differences between the
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treatment groups were assessed by one-way ANOVA using SPSS 19.0 (IBM, USA). A
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(GraphPad Software, Inc., USA) was used for Graphical illustrations.
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3. Results
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3.1. Effects of BPA, BPF and BPAF on body weight, liver weight and epiWAT
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weight
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Compared to the control group, the birth date and litter size did not significantly
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differ between the treatment groups. However, a significant increase in body weight
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and liver weight in mice exposed to BPA was observed (Fig.2A-B). BPA exposure
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resulted in significantly higher body weight gain in male offspring compared to the
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control group (Fig.2D). No significant changes in epididymal white adipose tissue
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(epiWAT) weight (Fig.2C) and the organ index of the liver (Fig.2E-F) occurred in the
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BPA treatment group. Similarly, perinatal exposure to BPF and BPAF had no effect on
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body weight, liver weight, and epiWAT weight, compared to the control group (Fig.2).
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3.2. Effects of BPA, BPF and BPAF on biochemical parameters and liver
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histopathology
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We explored the intrinsic changes in liver tissue through changes in liver weight,
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biochemical parameters, and liver histopathology. BPA and BPF exposure resulted in
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significantly increased TG levels compared, whilst BPAF exposure had no effect
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(Fig.3A). Significant changes in the liver T-Cho contents were observed in all
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treatment groups (Fig.3B). The activity of ALT and AST in the serum were measured
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to assess hepatic injury. ALT activity significantly increased following BPA exposure
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(Fig.3C) whilst no changes in AST activity were observed in the treatment groups
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ACCEPTED MANUSCRIPT (Fig.3D). These results demonstrate that lipid accumulation and liver damage were
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induced following perinatal exposure to BPA. This was further confirmed by
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histological analysis (Fig.3E) in which BPA and BPF exposure promoted lipid
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accumulation. Severe hepatocyte expansion in the BPA treatment group was also
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observed.
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3.3. Effects of BPA, BPF and BPAF on liver oxidative damage
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The activities of SOD, CAT and GSH-Px and the levels of GSH and MDA in the
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liver tissue are shown in Fig.4. In the BPA treatment group, the activity of SOD, CAT
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and GSH-Px, and the levels of GSH significantly decreased, whilst the levels of MDA
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increased compared to the control group (Fig.4A-E). Moreover, BPF exposure
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significantly reduced the activity of both CAT and GSH (Fig.4B, D). However, SOD,
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CAT and GSH-Px activity and the levels of GSH and MDA did not statistically differ
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between the BPAF treatment and control groups (Fig.4A-E).
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The relative expression levels of Sod1, Sod2, Gpx1, Gpx2 and Cat, the
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Nrf2-HO-1 pathway (Nrf2 and HO-1) and cell growth and apoptosis related genes
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(GADD45β) were detected by qRT-PCR (Fig.5). BPA exposure significantly reduced
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the relative expression of Sod1, Sod2, Gpx2 and Cat. Meanwhile, the relative
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expression of Nrf2 and HO-1 significantly increased in the BPA treatment group. The
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relative expression of Cat decreased in the BPF treatment group, whilst the expression
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of Sod1 significantly increased in the BPAF treatment group. These results suggested
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that perinatal exposure to both BPA and BPF led to oxidative stress in the livers of
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male mouse offspring.
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3.4. Effects of BPA, BPF and BPAF on liver metabolomics Representative 600 MHz 1H NMR spectra of the liver samples are shown in
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Figure S1. Chemical shift assignments of the metabolites have been summarized in
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previous studies (Yan et al., 2018). The 3D PCA score plots showed good separation
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in all treatment groups (Fig.6A). This indicates that the liver metabolic profiles were
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disturbed following BPA, BPF and BPAF exposure. Following this, PLS-DA and
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permutation plot models were used to identify the differential metabolites. PLS-DA
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score plots revealed significant differences between BPA, BPF and BPAF treatment
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groups compared to the control group (Fig.B-D). Moreover, 14 metabolites were
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found to be of significantly altered abundance across the three treatment groups (Fig.7;
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Table S2). Heatmaps of the metabolites were constructed to represent the clustering
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between individual samples. The results showed that 14 metabolites could be
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clustered into 2 types (Fig.7A). Specifically, the abundance of lipid, choline,
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phosphocholine (PC) and glycogen significantly increased with BPA exposure, whilst
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the levels of glycerophosphocholine (GPC), taurine, glutamate, glutamine, α-glucose,
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β-glucose, leucine, glycine and inosine decreased. In the BPF group, the relative
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levels of lipids and glycogen significantly increased, whilst the levels of PC,
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glutamate, α-glucose, β-glucose, isoleucine, and inosine significantly decreased. In
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the BPAF treatment group, the relative levels of β-glucose and inosine were
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significantly reduced, whilst higher levels of glycogen were observed (Fig.7B).
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4. Discussion
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ACCEPTED MANUSCRIPT In recent years, the widespread use of bisphenols (BPs) has made them
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ubiquitous in the natural environment (Liao et al., 2012), and the exposure to BPs
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through daily diet, skin contact and other means has increased (Hessel et al., 2016).
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BPA as the most representative BP has been shown to disrupt the endocrine system
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(Saal and Claude, 2005). Long-term exposure to BPA increases the risk of liver
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disorders (Nakagawa and Tayama, 2000), diabetes (Alonso-Magdalena et al., 2011)
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and cancer (Yang et al., 2009). Exposure to BPA analogs including BPF and BPAF
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also lead to adverse toxic effects (Park et al., 2018; Siracusa et al., 2018). However,
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studies on the toxicological properties of the early exposure to BPF and BPAF are
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sparse. In this study, we assessed the effects of perinatal BPA, BPF and BPAF
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exposure on systemic oxidative damage and metabolic disorders in male mouse
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offspring.
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We found that the body weight and liver weight of offspring mice significantly
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increased with BPA exposure. Subsequently, we examined the TG and T-Cho content
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in the liver, in addition to the activity of ALT and AST in the plasma to evaluate liver
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damage (Gordon et al., 2005). Compared to the control group, TG levels significantly
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increased with BPA and BPF exposure. In addition, BPA exposure significantly
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increased the activity of ALT. No such changes were observed following exposure to
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BPAF. Histopathological analysis confirmed these findings in which exposure to BPA
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and BPF resulted in lipid accumulation in the liver. Severe hepatocyte expansion was
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also observed in the BPA treatment group.
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oxidative damage in the liver (Li et al., 2016; Li et al., 2015; Liu et al., 2016). We
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evaluated the perinatal exposure to BPA, BPF, and BPAF on oxidative stress in the
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livers of the offspring. SOD, CAT and GSH-Px are common biomarkers associated
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with oxidative stress (Zhang et al., 2017). BPA exposure significantly reduced the
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activities of these antioxidant enzymes. CAT activity was significantly reduced in the
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BPF treatment group. As a product of lipid peroxidation, the levels of MDA reflect
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the degree of oxidative damage (Gupta et al., 2009). In the BPA treatment group, the
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levels of MDA significantly increased suggesting that the exposure to BPA resulted in
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lipid peroxidation and oxidative damage in the mouse livers. In addition, the
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overexpression of MDA influences the cellular structure and function (Marnett, 2002).
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BPA exposure reduced the levels of GSH and altered the relative expression of the
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oxidative damage-related genes Sod1, Sod2, Gpx2, and Cat. In particular, we found
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that the exposure to BPA significantly influenced the relative expression of the
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nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Nrf2
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is a major transcription factor that regulates the cellular defense against oxidative
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stress (Dayoub et al., 2011). Under oxidative stress, Nrf2 is translocated to the nucleus
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and activates the transcription of HO-1 (Edwards, 2007). As an endogenous
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cytoprotective enzyme, the relative expression of HO-1 significantly increased with
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oxidative stress, which would be damaging to living cells (Tang et al., 2005).
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However, the relative expression of GADD45β which is responsible for the regulation
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of cell growth and apoptosis showed no significant changes across the treatment
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relative expression of Sod1, Sod2, Cat and Gpx2 significantly decreased following
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BPA exposure, suggesting damage to the antioxidant defense system. In addition,
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BPA exposure decreased the levels of MDA and increased liver lipid peroxidation.
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These findings collectively demonstrate that BPA exposure induces oxidative liver
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damage. The differential exposure to BPF resulted in significantly decreased CAT
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activity and expression, suggesting a loss of antioxidant defenses. Exposure to BPAF
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decreased the relative expression of Sod1 suggesting that it does not perturb the
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antioxidant defense system.
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We further explored the impact of perinatal exposure to BPA, BPF, and BPAF
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on the liver metabolic profiles of the male mice. We identified significant changes in
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14 metabolites following BPA, BPF, and BPAF exposure, including those involved in
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lipid and glucose metabolism, amino acid metabolism, and glutamine and glutamate
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metabolism. The relative levels of 13 metabolites changed following BPA exposure,
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most notably increases in lipid, choline and PC, further indicating lipid accumulation
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following BPA perinatal exposure. Previous studies have demonstrated the
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development of liver oxidative damage in response to lipid metabolism disorders (Lv
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et al., 2017). Unlike the BPA treatment group, BPF and BPAF exposure led to fewer
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metabolic changes (8 and 2 metabolites, respectively). BPAF exposure caused fewer
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metabolite changes than BPA and BPF. However, β-glucose and glycogen levels
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significantly changed across the treatment groups, suggesting that BPA, BPF and
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BPAF exposure influenced liver glucose metabolism in the mice.
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toxic effects on male mouse offspring than BPA and BPF exposure, consistent with
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previous findings (Nakano et al., 2016). However, BPAF at low concentrations can
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induce oxidative damage to purine and pyrimidine bases (Mokra et al., 2018). To-date,
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few studies have assessed the toxic effects of perinatal exposure to BPF and BPAF on
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mouse offspring. Further systematic research in this area is now required.
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5. Conclusions
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In this study, we evaluated the effects of perinatal exposure to BPA, BPF and
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BPAF on liver function in male mouse offspring. The results showed that the perinatal
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exposure to BPA could induce oxidative damage and disrupt normal metabolic
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profiles in the liver. Moreover, exposure to BPF affected the liver antioxidant defense
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system and led to changes in the levels of 8 metabolites involved in lipid, glucose and
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amino acid metabolism. Exposure to BPAF had no negative effects on the oxidative
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system but altered the levels of β-glucose and glycogen. The underlying mechanisms
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controlling these effects now warrant further investigations.
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Acknowledgments
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We gratefully acknowledge the financial support from National Key Research and
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Development Program of China (2016YFD0200202), and the Young Elite Scientists
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Sponsorship Program by CAST.
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exposure affects liver function in mice involving oxidative damage. Regulatory
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Toxicology & Pharmacology Rtp 92, 138.
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Fig.1. Experimental design. Pregnant mice (F0) were orally gavaged with corn oil or BPA, BPF and BPAF (100 ng/g bw/day) corn oil solutions from gestational day (GD) 7 to postnatal day (PND) 21. At 13 weeks of age, the effects on
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oxidative damage and liver metabolomics in male mouse offspring (F1) were studied.
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Fig.2. Effects of BPA, BPF and BPAF on body weight, liver weight and epiWAT weight. A: Body weight. B: Liver weight. C: epiWAT weight. D: Body weight gain. E: %Liver weight/body weight. F: %EpiWAT weight/body weight. Data are
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Fig.3. Effect of BPA, BPF and BPAF on biochemical parameters and liver histopathology. A: Liver triglycerides (TG)
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contents. B: Total cholesterol (T-Cho) contents. C: Serum alanine aminotransferase (ALT) activities. D: Aspartate aminotransferase (AST) activities. E: Representative images of H&E staining of hepatic sections (100× and 400×, respectively) from different groups. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 compared with the control treatment (CK) group (n = 6 mouse per group).
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Fig.4. Effects of BPA, BPF and BPAF on hepatic oxidative stress levels. A: Liver superoxide dismutase (SOD) activities. B: Liver catalase (CAT) activities. C: Liver glutathione peroxidase (GSH-Px) activities. D: Liver glutathione levels. E: Liver malondialdehyde (MDA) levels. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 compared with the control
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Fig.5. Relative mRNA expression of several genes. A: Oxidative damage related genes (Sod1, Sod2, Gpx1, Gpx2 and Cat), B: Nrf2-HO-1 pathway (Nrf2 and HO-1). C: Cell growth and apoptosis related gene (GADD45β). Data are expressed as
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Fig.6. The 3D PCA score plot for all groups and the PLS-DA score plots for every two groups. A: The 3D PCA score plot of liver metabolites for all groups. B: The PLS-DA score plots of liver metabolites between BPA and control treatment groups. C: The PLS-DA score plots of liver metabolites between BPF and control treatment groups. D: The PLS-DA score plots of
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liver metabolites between BPAF and control treatment groups. (n = 6 mouse per group)
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Fig.7. Heatmap generated via MetaboAnalyst 4.0 and significantly changed metabolites. A: Heatmap; B: 14 significantly changed metabolites, including lipid, choline, PC, GPC, taurine, glutamate, glutamine, alpha-glucose, beta-glucose, glycogen, leucine, glycine, isoleucine and inosine. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 compared with the control treatment (CK) group (n = 6 mouse per group).
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Highlights: (1) Oxidative damage and metabolism disruption were induced in the liver of male mouse offspring with BPA exposure.
metabolic profiles of the liver of male mouse offspring.
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(2) BPF exposure could affect the antioxidant defense system and disrupt the
(3) BPAF exposure has no negative impact on oxidative damage, but it disrupts
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(4) Perinatal exposure to BPA, BPF and BPAF have different effects on liver
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oxidative damage and metabolic disorder in male mouse offspring.
S0269-7491(18)35272-2
DOI:
https://doi.org/10.1016/j.envpol.2019.01.116
Reference:
ENPO 12155
To appear in:
Environmental Pollution
Received Date: 22 November 2018 Revised Date:
29 January 2019
Accepted Date: 29 January 2019
Please cite this article as: Meng, Z., Tian, S., Yan, J., Jia, M., Yan, S., Li, R., Zhang, R., Zhu, W., Zhou, Z., Effects of perinatal exposure to BPA, BPF and BPAF on liver function in male mouse offspring involving in oxidative damage and metabolic disorder, Environmental Pollution (2019), doi: https:// doi.org/10.1016/j.envpol.2019.01.116. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effects of perinatal exposure to BPA, BPF and BPAF on liver
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function in male mouse offspring involving in oxidative damage and
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metabolic disorder
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Zhiyuan Meng a, Sinuo Tian a, Jin Yan a, Ming Jia a, Sen Yan a, Ruisheng Li a, Renke
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Zhang a, Wentao Zhu a *, Zhiqiang Zhou a
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a
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Department of Applied Chemistry, China Agricultural University, Beijing 100193,
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China
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Beijing Advanced Innovation Center for Food Nutrition and Human Health,
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* Corresponding author: Yuanmingyuan west road 2, Beijing 100193, PR China.
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E-mail address: [email protected]. (W. Zhu)
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ACCEPTED MANUSCRIPT Abstract: Bisphenols (BPs) are common environmental pollutants that are ubiquitous
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in the natural environment and can affect human health. In this study, we explored the
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effects of perinatal exposure to BPA, BPF and BPAF on liver function involving in
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oxidative damage and metabolic disorders in male mouse offspring. We found that
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BPA exposure impairs the antioxidant defense system, increases lipid peroxidation,
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and causes oxidative damage in the liver. Furthermore, the levels of 13 metabolites
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were significantly altered following BPA exposure. We found that BPF exposure
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significantly increased the expression and activity of CAT, suggesting disturbances in
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the antioxidant defense system. Moreover, BPF exposure led to metabolic disorders in
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the liver due to changes in the levels of 8 key metabolites. Exposure to BPAF caused
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no negative effects on oxidative damage, but altered the levels of β-glucose and
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glycogen. In summary, perinatal exposure to BPA, BPF and BPAF differentially
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influence oxidative damage and metabolic disorders in the livers of male mouse
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offspring. The impact of early life exposure to BPs now warrants future
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investigations.
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Capsule: Perinatal exposure to BPA, BPF and BPAF differentially influence oxidative
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damage, metabolic disorders, and subsequent liver function in male mouse offspring.
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Keywords: bisphenol A; bisphenol F; bisphenol AF; oxidative damage; metabolic
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disorder.
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1. Introduction Bisphenols (BPs) are important chemical substances that are widely used in the
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synthesis of plastics, particularly polycarbonates and epoxy resins. As the most
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commonly used bisphenol compound, bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)
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propane) is used in the production of plastic bottles, food plastic bags, medical
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materials, thermal paper and other products (Geens et al., 2012; Michalowicz, 2014).
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In 2015, the value of BPA used for the production of plastic monomers and
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plasticizers exceeded six billion pounds, making it one of the highest-volume
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chemicals worldwide. Due to its use in industrial applications, BPA enters the
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environment through various channels, leading to environmental risks. BPA has been
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reported to be distributed in water, soil, sediment, fish and foodstuffs, with the content
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of BPA reported as 0.08-0.47 µg/L in the surface waters of North America and Europe
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(Klecka et al., 2009). In China, the content of BPA is 0.04-0.64 µg/L in the Pearl
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River water and 0.88 µg/L in Guangzhou city water (Zhang and Zeng, 2016). The
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levels of BPA in fish liver and muscle tissue were also reported as 2-75 µg/kg and
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1-11 g/kg, respectively (Mita et al., 2011). In recent years due to increased toxicology
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and population epidemiology studies, the health risks of BPA have attracted
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widespread attention from both regulators and consumers. In the United States, the
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European Union, and other countries, the use of BPA is prohibited in food packaging
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products such as bottles (Michalowicz, 2014). Alternatives to BPA including
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bisphenol F (BPF, bis (4-hydroxyphenyl) methane) and bisphenol AF (BPAF,
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4,4’-(Hexafluoroisopropylidene) diphenol) have been increasingly used in industrial
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applications. Subsequently, BPF and BPAF have been detected in vegetables, meat,
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and seafood (Chen et al., 2016; Liao and Kannan, 2011, 2013). Importantly, humans can contact and absorb BPs from the natural environment.
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The average concentration of BPA detected in the urine samples of Belgian adults was
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3.9 ng/mL (Pirard et al., 2012). BPA was also detected in the urine of pregnant women
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during the first and second trimester (1.39 ng / mL and 1.27 ng / mL, respectively)
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(Chiu et al., 2017). Similarly to BPA, BPF and BPAF have been detected in the blood
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and adipose tissue of humans (Calafat et al., 2009; Mokra et al., 2018; Yang et al.,
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2012). The ability of BPA to disrupt the endocrine system has also been reported in
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numerous studies (Vandenberg et al., 2016). Similarly, adverse effects on the male
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reproductive system (Karnam et al., 2014) and thyroid function (Wang et al., 2015)
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have been documented. In addition, long-term exposure to BPA has been suggested to
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induce hypertension, diabetes, insulin resistance, obesity and heart disease (Cabaton
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et al., 2011; Sengupta et al., 2013; Shankar et al., 2012). Recent studies document that
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BPF and BPAF lead to adverse health effects and at low concentrations and can
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induce DNA damage (Mokra et al., 2018). Perinatal exposure to BPAF has also been
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shown to disrupt glycolipid homeostasis in the liver of female mouse offspring (Meng
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et al., 2018).
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Oxidative damage is a specific manifestation of environmental pollution that
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causes harm to humans and other organisms. The damage occurs as a result of
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excessive reactive oxygen species (ROS) due to an imbalance of oxidizing substances
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and the antioxidant system (Hirooka et al., 2010). Studies have shown that BPA
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Elobeid, 2012). Moreover, exposure to BPF causes oxidative stress in the immune
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system of juvenile common carp (Cyprinus carpio) (Qiu et al., 2018). In vitro studies
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have shown that BPAF has a negative impact on oocyte maturation in mice through
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oxidative stress and DNA damage (Ding et al., 2017). Studies on the association of
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oxidative damage and metabolic disorders induced by BPA, BPF and BPAF exposure
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have not been reported.
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In this study, we assessed the effects of BPA, BPF and BPAF on the liver
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function of male mouse offspring, including oxidative damage and metabolic
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disorders, following perinatal exposure. Specifically, we examined changes in the
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activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase
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(GSH-Px), changes in the levels of glutathione (GSH) and malondialdehyde (MDA),
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and alterations in the relative expression of oxidative stress-related genes including
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Sod1, Sod2, Cat, Gpx1, Gpx2, Nrf2, HO-1, and GADD45β in the liver tissue of male
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mouse offspring. In addition, the liver metabolic profiles of the offspring were
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assessed using 1H NMR. The results of this study provide a theoretical basis for
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further comprehensive analysis of the health risks that result from perinatal exposure
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to BPs.
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2. Materials and Methods
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2.1. Animals and treatments
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A total of 32 primigravida pregnant ICR mice were obtained from the Peking
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University Health Science Center (Beijing, China). Mice were maintained in
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and a normal diet (Keao Xieli Feed Co., Ltd. Beijing, China). Based on the mother's
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weight, BPA, BPF, and BPAF corn oil solutions were orally administered to pregnant
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mice (8 pregnant mice per group) at a dose of 100 ng/g bw/day from the 7th day of
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pregnancy to the 21st day after delivery. Control mice received only corn oil treatment.
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Each dam was culled to six male cubs the day after birth. Male offspring in each
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treatment group were provided a normal diet for 10 weeks after weaning at day 21.
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The specific experimental design is shown in Fig.1. Body weights were measured
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once a week and liver tissue and serum samples were collected at the end of this study.
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Samples were stored at −80℃ for further analysis.
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2.2. Analysis of biochemical parameters
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Serum samples from mice offspring at 13 weeks were collected for the detection
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of ALT and AST activity using commercial assay kits. The contents of triglyceride
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(TG) and total cholesterol (T-Cho) in the liver samples was also quantified. For the
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assessment of oxidative parameters, liver tissue (100 mg) was homogenized in 900 µL
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of saline and precipitates were collected by centrifugation at 3000 rpm for 12 min.
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Supernatants were collected for the assessment of SOD, CAT and GSH-Px activity
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and the levels of MDA and GSH using commercial analysis kits (Nanjing Jiancheng
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Bioengineering Institute, Nanjing, China) according to the manufacturer’s
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instructions.
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2.3. Histopathological analysis
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Liver sections from 13 week old mice were embedded in paraffin for histological
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examination. Sections of 5-6 µm were stained with hematoxylin and eosin (H & E)
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and analyzed on an Olympus microscope.
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2.4. Analysis of mRNA expression Liver samples were frozen in liquid nitrogen and grounded. TRIzol reagent
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(Tiangen Biotech Co., LTD., Beijing, China) was used to extract total RNA from the
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liver samples. cDNA was synthesized from total RNA (1.5 µg) using Fast Quant RT
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Kit (Tiangen Biotech Co., LTD., Beijing, China). qRT-PCR was performed using
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SuperReal PreMix Plus (SYBR Green) (Qiagen, China) and the Bio-Rad CFX 96
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PCR system (Bio-Rad, USA), respectively. Gene expression was normalized to
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β-actin. Primers were obtained from Sangon Biotech (Shanghai, China) and are listed
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in Table S1.
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2.5. Liver metabolomics analysis
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The preparation of liver samples for 1H NRM analysis was performed as
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previously described (Meng et al., 2018) and detailed in supplementary information.
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1
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Billerica, MA, USA). Multivariate statistical analysis was performed using SIMCA P
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(Version 11, Umetrics, Sweden). 1H NMR parameters and multivariate statistical
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analysis were performed as previously described and outlined in Supporting
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information S2 (Zhang et al., 2015; Wang et al., 2018).
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2.6. Statistical analysis
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All values are expressed as the mean ± SD. Statistical differences between the
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treatment groups were assessed by one-way ANOVA using SPSS 19.0 (IBM, USA). A
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(GraphPad Software, Inc., USA) was used for Graphical illustrations.
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3. Results
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3.1. Effects of BPA, BPF and BPAF on body weight, liver weight and epiWAT
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weight
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Compared to the control group, the birth date and litter size did not significantly
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differ between the treatment groups. However, a significant increase in body weight
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and liver weight in mice exposed to BPA was observed (Fig.2A-B). BPA exposure
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resulted in significantly higher body weight gain in male offspring compared to the
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control group (Fig.2D). No significant changes in epididymal white adipose tissue
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(epiWAT) weight (Fig.2C) and the organ index of the liver (Fig.2E-F) occurred in the
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BPA treatment group. Similarly, perinatal exposure to BPF and BPAF had no effect on
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body weight, liver weight, and epiWAT weight, compared to the control group (Fig.2).
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3.2. Effects of BPA, BPF and BPAF on biochemical parameters and liver
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histopathology
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We explored the intrinsic changes in liver tissue through changes in liver weight,
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biochemical parameters, and liver histopathology. BPA and BPF exposure resulted in
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significantly increased TG levels compared, whilst BPAF exposure had no effect
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(Fig.3A). Significant changes in the liver T-Cho contents were observed in all
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treatment groups (Fig.3B). The activity of ALT and AST in the serum were measured
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to assess hepatic injury. ALT activity significantly increased following BPA exposure
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(Fig.3C) whilst no changes in AST activity were observed in the treatment groups
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induced following perinatal exposure to BPA. This was further confirmed by
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histological analysis (Fig.3E) in which BPA and BPF exposure promoted lipid
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accumulation. Severe hepatocyte expansion in the BPA treatment group was also
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observed.
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3.3. Effects of BPA, BPF and BPAF on liver oxidative damage
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The activities of SOD, CAT and GSH-Px and the levels of GSH and MDA in the
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liver tissue are shown in Fig.4. In the BPA treatment group, the activity of SOD, CAT
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and GSH-Px, and the levels of GSH significantly decreased, whilst the levels of MDA
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increased compared to the control group (Fig.4A-E). Moreover, BPF exposure
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significantly reduced the activity of both CAT and GSH (Fig.4B, D). However, SOD,
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CAT and GSH-Px activity and the levels of GSH and MDA did not statistically differ
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between the BPAF treatment and control groups (Fig.4A-E).
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The relative expression levels of Sod1, Sod2, Gpx1, Gpx2 and Cat, the
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Nrf2-HO-1 pathway (Nrf2 and HO-1) and cell growth and apoptosis related genes
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(GADD45β) were detected by qRT-PCR (Fig.5). BPA exposure significantly reduced
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the relative expression of Sod1, Sod2, Gpx2 and Cat. Meanwhile, the relative
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expression of Nrf2 and HO-1 significantly increased in the BPA treatment group. The
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relative expression of Cat decreased in the BPF treatment group, whilst the expression
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of Sod1 significantly increased in the BPAF treatment group. These results suggested
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that perinatal exposure to both BPA and BPF led to oxidative stress in the livers of
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male mouse offspring.
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3.4. Effects of BPA, BPF and BPAF on liver metabolomics Representative 600 MHz 1H NMR spectra of the liver samples are shown in
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Figure S1. Chemical shift assignments of the metabolites have been summarized in
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previous studies (Yan et al., 2018). The 3D PCA score plots showed good separation
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in all treatment groups (Fig.6A). This indicates that the liver metabolic profiles were
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disturbed following BPA, BPF and BPAF exposure. Following this, PLS-DA and
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permutation plot models were used to identify the differential metabolites. PLS-DA
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score plots revealed significant differences between BPA, BPF and BPAF treatment
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groups compared to the control group (Fig.B-D). Moreover, 14 metabolites were
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found to be of significantly altered abundance across the three treatment groups (Fig.7;
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Table S2). Heatmaps of the metabolites were constructed to represent the clustering
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between individual samples. The results showed that 14 metabolites could be
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clustered into 2 types (Fig.7A). Specifically, the abundance of lipid, choline,
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phosphocholine (PC) and glycogen significantly increased with BPA exposure, whilst
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the levels of glycerophosphocholine (GPC), taurine, glutamate, glutamine, α-glucose,
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β-glucose, leucine, glycine and inosine decreased. In the BPF group, the relative
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levels of lipids and glycogen significantly increased, whilst the levels of PC,
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glutamate, α-glucose, β-glucose, isoleucine, and inosine significantly decreased. In
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the BPAF treatment group, the relative levels of β-glucose and inosine were
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significantly reduced, whilst higher levels of glycogen were observed (Fig.7B).
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4. Discussion
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ACCEPTED MANUSCRIPT In recent years, the widespread use of bisphenols (BPs) has made them
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ubiquitous in the natural environment (Liao et al., 2012), and the exposure to BPs
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through daily diet, skin contact and other means has increased (Hessel et al., 2016).
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BPA as the most representative BP has been shown to disrupt the endocrine system
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(Saal and Claude, 2005). Long-term exposure to BPA increases the risk of liver
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disorders (Nakagawa and Tayama, 2000), diabetes (Alonso-Magdalena et al., 2011)
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and cancer (Yang et al., 2009). Exposure to BPA analogs including BPF and BPAF
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also lead to adverse toxic effects (Park et al., 2018; Siracusa et al., 2018). However,
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studies on the toxicological properties of the early exposure to BPF and BPAF are
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sparse. In this study, we assessed the effects of perinatal BPA, BPF and BPAF
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exposure on systemic oxidative damage and metabolic disorders in male mouse
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offspring.
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We found that the body weight and liver weight of offspring mice significantly
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increased with BPA exposure. Subsequently, we examined the TG and T-Cho content
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in the liver, in addition to the activity of ALT and AST in the plasma to evaluate liver
225
damage (Gordon et al., 2005). Compared to the control group, TG levels significantly
226
increased with BPA and BPF exposure. In addition, BPA exposure significantly
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increased the activity of ALT. No such changes were observed following exposure to
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BPAF. Histopathological analysis confirmed these findings in which exposure to BPA
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and BPF resulted in lipid accumulation in the liver. Severe hepatocyte expansion was
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also observed in the BPA treatment group.
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oxidative damage in the liver (Li et al., 2016; Li et al., 2015; Liu et al., 2016). We
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evaluated the perinatal exposure to BPA, BPF, and BPAF on oxidative stress in the
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livers of the offspring. SOD, CAT and GSH-Px are common biomarkers associated
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with oxidative stress (Zhang et al., 2017). BPA exposure significantly reduced the
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activities of these antioxidant enzymes. CAT activity was significantly reduced in the
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BPF treatment group. As a product of lipid peroxidation, the levels of MDA reflect
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the degree of oxidative damage (Gupta et al., 2009). In the BPA treatment group, the
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levels of MDA significantly increased suggesting that the exposure to BPA resulted in
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lipid peroxidation and oxidative damage in the mouse livers. In addition, the
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overexpression of MDA influences the cellular structure and function (Marnett, 2002).
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BPA exposure reduced the levels of GSH and altered the relative expression of the
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oxidative damage-related genes Sod1, Sod2, Gpx2, and Cat. In particular, we found
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that the exposure to BPA significantly influenced the relative expression of the
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nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Nrf2
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is a major transcription factor that regulates the cellular defense against oxidative
247
stress (Dayoub et al., 2011). Under oxidative stress, Nrf2 is translocated to the nucleus
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and activates the transcription of HO-1 (Edwards, 2007). As an endogenous
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cytoprotective enzyme, the relative expression of HO-1 significantly increased with
250
oxidative stress, which would be damaging to living cells (Tang et al., 2005).
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However, the relative expression of GADD45β which is responsible for the regulation
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of cell growth and apoptosis showed no significant changes across the treatment
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ACCEPTED MANUSCRIPT groups. The activities of SOD, CAT and GSH-Px were significantly lower and the
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relative expression of Sod1, Sod2, Cat and Gpx2 significantly decreased following
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BPA exposure, suggesting damage to the antioxidant defense system. In addition,
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BPA exposure decreased the levels of MDA and increased liver lipid peroxidation.
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These findings collectively demonstrate that BPA exposure induces oxidative liver
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damage. The differential exposure to BPF resulted in significantly decreased CAT
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activity and expression, suggesting a loss of antioxidant defenses. Exposure to BPAF
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decreased the relative expression of Sod1 suggesting that it does not perturb the
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antioxidant defense system.
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We further explored the impact of perinatal exposure to BPA, BPF, and BPAF
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on the liver metabolic profiles of the male mice. We identified significant changes in
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14 metabolites following BPA, BPF, and BPAF exposure, including those involved in
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lipid and glucose metabolism, amino acid metabolism, and glutamine and glutamate
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metabolism. The relative levels of 13 metabolites changed following BPA exposure,
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most notably increases in lipid, choline and PC, further indicating lipid accumulation
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following BPA perinatal exposure. Previous studies have demonstrated the
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development of liver oxidative damage in response to lipid metabolism disorders (Lv
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et al., 2017). Unlike the BPA treatment group, BPF and BPAF exposure led to fewer
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metabolic changes (8 and 2 metabolites, respectively). BPAF exposure caused fewer
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metabolite changes than BPA and BPF. However, β-glucose and glycogen levels
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significantly changed across the treatment groups, suggesting that BPA, BPF and
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BPAF exposure influenced liver glucose metabolism in the mice.
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toxic effects on male mouse offspring than BPA and BPF exposure, consistent with
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previous findings (Nakano et al., 2016). However, BPAF at low concentrations can
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induce oxidative damage to purine and pyrimidine bases (Mokra et al., 2018). To-date,
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few studies have assessed the toxic effects of perinatal exposure to BPF and BPAF on
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mouse offspring. Further systematic research in this area is now required.
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5. Conclusions
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In this study, we evaluated the effects of perinatal exposure to BPA, BPF and
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BPAF on liver function in male mouse offspring. The results showed that the perinatal
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exposure to BPA could induce oxidative damage and disrupt normal metabolic
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profiles in the liver. Moreover, exposure to BPF affected the liver antioxidant defense
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system and led to changes in the levels of 8 metabolites involved in lipid, glucose and
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amino acid metabolism. Exposure to BPAF had no negative effects on the oxidative
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system but altered the levels of β-glucose and glycogen. The underlying mechanisms
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controlling these effects now warrant further investigations.
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Acknowledgments
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We gratefully acknowledge the financial support from National Key Research and
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Development Program of China (2016YFD0200202), and the Young Elite Scientists
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Sponsorship Program by CAST.
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Fig.1. Experimental design. Pregnant mice (F0) were orally gavaged with corn oil or BPA, BPF and BPAF (100 ng/g bw/day) corn oil solutions from gestational day (GD) 7 to postnatal day (PND) 21. At 13 weeks of age, the effects on
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Fig.2. Effects of BPA, BPF and BPAF on body weight, liver weight and epiWAT weight. A: Body weight. B: Liver weight. C: epiWAT weight. D: Body weight gain. E: %Liver weight/body weight. F: %EpiWAT weight/body weight. Data are
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Fig.3. Effect of BPA, BPF and BPAF on biochemical parameters and liver histopathology. A: Liver triglycerides (TG)
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Fig.4. Effects of BPA, BPF and BPAF on hepatic oxidative stress levels. A: Liver superoxide dismutase (SOD) activities. B: Liver catalase (CAT) activities. C: Liver glutathione peroxidase (GSH-Px) activities. D: Liver glutathione levels. E: Liver malondialdehyde (MDA) levels. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 compared with the control
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treatment (CK) group (n = 6 mouse per group).
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Fig.5. Relative mRNA expression of several genes. A: Oxidative damage related genes (Sod1, Sod2, Gpx1, Gpx2 and Cat), B: Nrf2-HO-1 pathway (Nrf2 and HO-1). C: Cell growth and apoptosis related gene (GADD45β). Data are expressed as
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mean ± SD. *p < 0.05, **p < 0.01 compared with the control treatment (CK) group (n = 6 mouse per group).
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Fig.6. The 3D PCA score plot for all groups and the PLS-DA score plots for every two groups. A: The 3D PCA score plot of liver metabolites for all groups. B: The PLS-DA score plots of liver metabolites between BPA and control treatment groups. C: The PLS-DA score plots of liver metabolites between BPF and control treatment groups. D: The PLS-DA score plots of
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liver metabolites between BPAF and control treatment groups. (n = 6 mouse per group)
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Fig.7. Heatmap generated via MetaboAnalyst 4.0 and significantly changed metabolites. A: Heatmap; B: 14 significantly changed metabolites, including lipid, choline, PC, GPC, taurine, glutamate, glutamine, alpha-glucose, beta-glucose, glycogen, leucine, glycine, isoleucine and inosine. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 compared with the control treatment (CK) group (n = 6 mouse per group).
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Highlights: (1) Oxidative damage and metabolism disruption were induced in the liver of male mouse offspring with BPA exposure.
metabolic profiles of the liver of male mouse offspring.
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(2) BPF exposure could affect the antioxidant defense system and disrupt the
(3) BPAF exposure has no negative impact on oxidative damage, but it disrupts
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glucose metabolism in the liver of male mouse offspring.
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(4) Perinatal exposure to BPA, BPF and BPAF have different effects on liver
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oxidative damage and metabolic disorder in male mouse offspring.