Chemicals

Methanol, acetone and hexane were HPLC-grade, obtained from Fisher Chemicals (Fair Lawn, NJ). Dimethyl sulfoxide (DMSO) was ACS-grade, obtained from AMRESCO. BHPF (>98%), E 2 (≥98%), OHT (≥98%), FULV (>98%) and 9-fluorenone (>98%) were purchased from Sigma-Aldrich (St Louis, MO). TAM (≥99%) was obtained from Aladdin Reagents (Shanghai, China). Phenol-d 5 (RING-D5,>98%) was purchased from Cambridge Isotope Laboratories (Andover, MA). β-Mercaptopropionic acid (>98%) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).

Identification of BHPF from a plastic bottle by NMR

Methanol leachates from the grated plastic of a ‘BPA-free’ bottle were isolated and purified by liquid chromatography with a C18 preparative column. The purified substance was used for 1H and 13C NMR analysis. 1H NMR and 13C NMR spectra were recorded at 400 and 600 MHz on a Bruker AVANCE 400 spectrometer and a Bruker AVANCE DRX-600 spectrometer, respectively.

Synthesis of deuterated BHPF

A mixture of 9-fluorenone, phenol-d 5 and β-mercaptopropionic acid was added to a 50 ml three-neck bottom flask equipped with a magnetic stirrer, reflux condenser and thermometer, and then concentrated sulphuric acid was slowly added dropwise with stirring. The mixture was heated at 55 °C for 4 h, and then washed several times with water and methanol, and dried. The dried powder was recrystallized from toluene, and deuterated BHPF was obtained. By analysis of this deuterated BHPF, it was identified as BHPF-d 7 (Supplementary Fig. 1a,b), which was used as a surrogate standard for the GC–MS analysis of BHPF.

Analysis of BHPF in water released from plastic bottles

Fifty two plastic bottles, including adult water bottles, children water bottles and sippy cups, and baby milk bottles, with volume in the range of 240–1,000 ml, were purchased in China and in foreign markets (Supplementary Table 1). The plastic bottles were filled with water heated to 60 °C, and allowed to sit for 6 h before the determination of BHPF. A water sample spiked with 20 ng of surrogate standard (BHPF-d 7 ) was extracted on a Phenomenex strata-x SPE cartridge (500 mg 6 ml−1). The cartridges were preconditioned with 20 ml of methanol and 8 ml of distilled water, and then rinsed with 5 ml of distilled water and 8 ml of methanol/water (1:20, v/v). After being dried under a flow of nitrogen, the cartridge was eluted with 8 ml of methanol. The extracts were then dried under a flow of nitrogen and derivatized with 100 μl BSTFA+1% TMCS (Regis Technologies, Inc., Morton Grove, IL) in acetone. After 30 min of reaction, 1 ml of water was added to the incubation mixtures, and they were extracted with n-hexane (1:1) three times. The extracts were evaporated to dryness under a gentle nitrogen stream and dissolved in 100 μl of n-hexane for GC–MS analysis (Agilent 7890A/5975C). An HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm film thickness; Agilent) was used to separate the target chemicals. A splitless injector was used and was maintained at 300 °C. The temperature program ranged from 100 to 300 °C at a rate of 15 °C min−1. The interface temperature, ion temperature and quadrupole temperature were maintained at 300, 230 and 150 °C, respectively. The carrier gas was helium supplied at a constant flow rate of 1 ml min−1. The injection volume was 1 μl. As shown in Supplementary Fig. 1b, O-bis(trimethylsilyl) derivatives were observed at m/z=494 and 501 for BHPF and BHPF-d 7 , respectively. The recoveries of BHPF and BHPF-d 7 in the spiked water samples were in the range of 80.8–103.5%, and the limit of detection was calculated as 0.1 ng l−1.

Analysis of BHPF in human serum

In China, college students usually undergo a physical examination before graduation. The volunteers in this study were senior students who were undergoing a physical examination in Beijing. Fifty male and 50 female healthy volunteers (mean age, 23.5±1.2 years) who habitually used plastic water bottles were randomly recruited to participate in this study in June 2015, after appropriate ethical approval was received from the Human Ethics Committee of the Peking University Peoples’ Hospital. Each participant provided informed consent and volunteered to give one 10-ml random sample of blood. Fasting blood was collected at Peking University Hospital Medical Center with vacutainer blood collection device (Chengwu Yongkang Medical Products Ltd., China) between 8 and 9 h. The blood collection device principally consisted of a steel needle with sharp ends, which linked the blood–vessel lumen with the glass vacutainer tube during blood collection, and a polypropylene auxiliary syringe for holding the blood, which had been verified to be free of BHPF contamination. The blood was collected directly into plain 10-ml glass vacutainer tubes and spun down 30 min later for serum preparation. The serum samples were stored in glass tubes at −80 °C until analysis. The samples were fortified with 5 ng of surrogate standard BHPF-d 7 , and the pH was adjusted to 6.5 with ammonium acetate. β-Glucuronidase/arylsulfatase (20 μl; Sigma-Aldrich, St Louis, MO) was added to the samples, and they were incubated at 37 °C for 20 h to allow deglucuronidation. After enzymatic hydrolysis, the samples were diluted and loaded onto a Phenomenex strata-x SPE cartridge (200 mg 3 ml−1) that had previously been conditioned with 6 ml of methanol and 3 ml of distilled water. The cartridge was rinsed with 3 ml of distilled water and 4 ml of methanol/water (1:20, v/v). After being dried under a flow of nitrogen, the cartridge was eluted with 4 ml of methanol. The eluate was evaporated to dryness, and dissolved in n-hexane for GC–MS analysis. All analytical procedures were checked for precision, reproducibility, blank contamination and linearity. Quality control was maintained by analysing a method blank (calf serum) and two spiked calf serum samples (piked with BHPF-d 7 singly or a mixture of BHPF-d 7 and undeuterated BHPF) along with every 12 samples. No BHPF was detected in the blank and single BHPF-d 7 spiked calf serum samples during the analytical procedures. The detection limit (0.1 ng ml−1) was based upon the criterion of the instrument having a signal-to-noise response of 3:1.

Yeast two-hybrid assay

The oestrogenic and anti-oestrogenic activities of BHPF were tested by a yeast two-hybrid assay with human oestrogen receptor α and the coactivator transcriptional intermediary factor 2 (refs 39, 40). Yeast cells (Saccharomyces cerevisiae Y190) expressing the human oestrogen receptor α (ERα) were grown overnight at 30 °C with vigorous shaking in synthetic defined medium lacking tryptophan and leucine. The overnight culture was then added to fresh medium and treated with test chemicals at 30 °C for 4 h. After the incubation, the treated cells were collected and digested with 1 mg ml−1 Zymolyase-20T (Seikagaku Kogyo Co., Ltd., Tokyo) at 37 °C for 20 min, and then 2-Nitrophenyl β-D-galactopyranoside was added to the lysate to a final concentration of 4 mg ml−1. After incubation at room temperature for 20 min, the yellow colour developed and 100 ml of 1 M Na 2 CO 3 were added to stop the reaction. Then, the yeast debris was removed by centrifugation and β-galactosidase activities were determined by measuring the absorbance of supernatant at 415 nm in a spectrophotometer. To determine whether BHPF possessed anti-oestrogenic activity, 1 nM of E 2 with the diluted BHPF was added to the medium. OHT was used as a positive control. All experiments were performed in triplicate. Sigmoidal dose–effect curves were calculated using the GraphPad Prism 4 software. The IC 50 values were calculated on the basis of the sigmoidal dose-effect curves.

Dual-luciferase reporter assays

Dual-luciferase reporter assays were carried out primarily according to the method described in a previous study41. In short, full-length sequences of human oestrogen receptor α and β were amplified by RT-PCR using mRNA from MCF-7 (Supplementary Table 3), and inserted into a pSVSPORT1 (Invitrogen); the resulting constructs were termed pSVhERα and pSVhERβ. A reporter plasmid containing ERE was constructed by inserting four tandem repeats of an ERE of Xenopus laevis vitellogenin A2 into a pGL4.23 vector (Promega), and was termed pGL4-ERE-luc. All of the plasmids constructed were confirmed by sequence analysis. The Renilla LUC control reporter construct pGL4.74-TK was purchased from Promega (Madison, WI). Human choriocarcinoma JEG-3 cells (ATCC No. HTB-36) were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and were cultured in minimal essential medium (MEM, Invitrogen, Carlsbad, CA) containing 2 mM L-glutamine, 0.1 mM minimal essential medium nonessential amino acid solution (Invitrogen/Thermo Fisher Scientific, Grand Island, NY), and 10% fetal calf serum (FCS) at 37 °C in a humidified atmosphere containing 5% CO 2 . The cells (3 × 104 cells per well) were seeded in 24-well plates 24 h before transfection. The cells were transfected with pGL4-ERE-luc (5 ng per well), pGL 4.74-TK (0.2 ng per well) and either pSVhERα (5 ng per well) or pSVhERβ (5 ng per well) using Lipofectamine reagent (Invitrogen). At 24 h after transfection, the compounds in DMSO were added to the cultures at a volume ratio (v/v) of 0.1%. The cells were continuously cultured in medium supplemented with 1% charcoal-stripped FCS for another 24 h and then harvested, and cell extracts were assayed for firefly luciferase activity. The assays were performed in quadruplicate, and the results were expressed as the fold induction of the control after normalization against Renilla luciferase activity. Sigmoidal dose-effect curves were calculated using the GraphPad Prism 4 software. The IC 50 values were calculated on the basis of the sigmoidal dose-effect curves. Finally, a WST-1 cell proliferation assay was performed to evaluate the cytotoxicity of BHPF using the cell proliferation reagent WST-1 (Dojindo, Mashiki, Japan). Aliquots (200 μl) of JEG-3 cells (1 × 104 cells per well) were seeded into 96 well-plates and precultured for 24 h. The cells were then treated with various concentrations of BHPF. After another 24 h, 20 μl of 5 mM WST-1 with 0.2 mM 1-Methoxy-5-methylphenazinium methylsulfate was added for 4 h before the absorbance at 490 nm was measured with a microplate reader (iMark, BioRad). The WST-1 cell proliferation assay was performed in quadruplicate.

Molecular docking

Scigress (Ultra Version 3.0.0, Fujitsu) was used for in silico molecular docking analysis42,43. The three-dimensional structures of the ligand-binding domain of human oestrogen receptor α, PDB IDs 1ERE, 3UU7, 3UUA, 3UUC and 3ERT, were downloaded from the Protein Data Bank website (http://www.rcsb.org.pdb) and used to evaluate the binding affinities of BHPF in the agonist and antagonist pockets of human oestrogen receptor α, respectively. The structures were refined, and all of the water molecules were removed from the protein, except for those that were important to the ligand-binding pocket composition, using Scigress-integrated procedures. The structures of BHPF and the original ligands were drawn, cleaned, and energy-optimized for molecular modulation. Automated docking of the flexible ligands into the flexible active sites was carried out using a genetic algorithm. The docking calculations were evaluated on a 0.25 Å grid in a 15 × 15 × 15 Å box containing the active site for the original ligand. The local search parameters included 300 maximum iterations at a rate of 0.06, and the procedure was set to run 30,000 generations with an initial population size of 50, an elitism of 7, a crossover of 0.8, a mutation of 0.2, and a convergence of 1.0.0. After automated docking, a geometric optimization calculation was performed for the human oestrogen receptor α-BHPF complex using augmented MM3 parameters.

Anti-uterotrophic assays using mice

All animal studies were approved by the Institutional Animal Care and Use Committee of Peking University, and were performed in accordance with the Guidelines for Animal Experiments of the university, which meet the ethical guidelines for experimental animals in China. Immature female CD-1 mice (20 days old) were obtained from Experimental Animal Tech Co. of Weitonglihua (Beijing, China). Mice with a maximal difference in body weight of 1 g were selected for the experiments and randomly assigned to either the treatment or control groups. The animals were housed five to a cage and acclimatized in a controlled environment with a temperature of 22±2 °C, a relative humidity between 40 and 60%, and artificial lighting in a 12 h/12 h light-dark cycle. The animals were fed ad libitum with a basic diet from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China), and drinking water was provided ad libitum. Before the experiments, the mice were randomly assigned to either the treatment or the control group (n=10). Mice treated with peanut oil were used as a vehicle control, and the chemicals were dissolved in peanut oil to the appropriate concentrations. The volume of vehicle or chemical solutions administered was adjusted daily based on body weight measured during the dosing period according to the volume-body weight ratio of 5 ml kg−1 bw. The treatment via oral gavage to each mouse was performed for 10 days beginning on PND 24. Mice in each cage were labelled by shaving hair on different parts of the body, and the oral gavage treatment was performed in turns for each group, whereby in each turn only one mouse in a group was treated. After the period of treatment, the mice were weighed and killed 24 h after the final treatment according to the sequence of the treatments, and the uteri were removed, blotted and weighed by one experimenter to reduce the risk of bias in the data collection. The relative uterine weight, that is, the ratio of uterine weight to final body weight, was calculated to evaluate the anti-uterotrophic activity of the chemicals.

Expression profiling and Q-RT-PCR

Immature female CD-1 mice were obtained from Experimental Animal Tech Co. of Weitonglihua (Beijing, China) and acclimatized in an experimental environment with a temperature of 22 °C±2 °C, relative humidity between 40 and 60%, and artificial lighting in a 12 h/12 h light-dark cycle. The animals were fed ad libitum with a basic diet from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China), and drinking water was provided ad libitum. The mice were treated with 50 mg kg−1 bw d−1 of BHPF or peanut oil via oral gavage for 3 days beginning on PND 21. On PND 24, mice of each group were weighed and killed by cervical dislocation. The uteri, ovaries and livers were collected for total RNA isolation. Total RNA was isolated using Trizol reagent (Invitrogen) and further purified by NucleoSpin RNA Clean-up (Macherey-Nagel, Germany). Fluorescence labelling of the RNA samples was performed with a Jingxin cRNA linear amplification and labelling kit (CapitalBio) according to the manufacturer’s instructions. The total RNA from each pool of tissue samples (a mixture from three animals) was used for microarray analysis. Cy3 dCTP was used to label the samples. The labelled products were added to SurePrint G3 mouse gene expression 8 × 60 K microarrays (Agilent) and incubated overnight at 42 °C. Following hybridization, the gene arrays were washed and scanned using an Agilent G2565CA Microarray Scanner. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control to monitor the quality of the target preparation. The data were processed with feature extraction software (GeneSpring GX v.11; Agilent). The raw signals were log-transformed and normalized with the percentile shift normalization method. All arrays met the minimum Agilent QA/QC standards. To verify the data from the gene array, 14 genes were chosen as candidates for Q-RT-PCR. Primers are listed in Supplementary Table 4. GAPDH was used as an endogenous control. Amplification was performed using SYBR Green PCR master mix (Applied Biosystems) according to the manufacturer’s instructions. The relative expression was quantified using the 2(−ΔΔCt) method. The Gene Ontology (GO) and KEGG pathway enrichment analyses were performed with DAVID (refs 40, 41). Sprr2a and sprr2b were selected as biomarker genes, and their expressions in the uteri of the mice (n=10) given BHPF at doses of 0, 9.26, 27.8, 83.3 and 250 mg kg−1 bw d−1 for 10 days were studied by Q-RT-PCR.

Subchronic and reproductive toxicity tests using mice

Immature CD-1 mice (20 days old) were obtained from Experimental Animal Tech Co. of Weitonglihua (Beijing, China) and acclimatized in an experimental environment with a temperature of 22 °C±2 °C, relative humidity between 40 and 60%, and artificial lighting in a 12 h/12 h light-dark cycle. The animals were fed ad libitum with a basic diet from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China), and drinking water was provided ad libitum. The animals were randomized into 6 groups (vehicle control group, 0.4, 2, 10 and 50 mg kg−1 BHPF groups, and 1.2 mg kg−1 TAM group) with 15 males and 25 females per group. The animals were housed five to a cage until the pairing date. The treatment via oral gavage of each mouse was performed once every 3 days beginning on PND 24. The chemicals were dissolved in peanut oil to the appropriate concentrations. The volume of vehicle or chemical solutions administered was adjusted daily based on body weight measured during the dosing period according to the volume-body weight ratio of 5 ml kg−1 bw. On PND 60, 10 females of each group were weighed and killed by cervical dislocation. The uteri, ovaries, livers, kidneys and spleens were weighed and collected for histological analysis. At the age of PND 77, males and females were paired. Each morning, the females were examined for the presence of vaginal plugs. Gestational day 1 was defined as the day on which a vaginal plug was found. In cases where pairing was unsuccessful, females were re-mated with proven males of the same group, except that females in the 1.2 mg kg−1 TAM group were re-mated with proven males of the control group. After a 7-day mating period, the males were weighed and killed, and organs were weighed and collected for further analysis. Cauda epididymal sperm was collected from each mouse and observed under a trinocular microscope (Olympus BX53) in physiological saline. A microscopic video was recorded and analysed for sperm quality evaluation. The treatment via oral gavage was continued for the females until day 19 of pregnancy. On post-partum days 0 and 4, the weights of the dams and pups were recorded and the pups were checked for abnormalities. On post-partum day 4, the dams were weighed and killed, and their organs were weighed and collected for further analysis. The ovaries and uteri were examined for the numbers of corpora lutea and implantation sites, respectively. Finally, the non-pregnant and nulliparous females were killed for histopathological analysis. For histological examination, organs and tissues were fixed in formalin fixation, embedded in paraffin, and sectioned serially at 5 μm. Slides were stained with hematoxylin and eosin (H&E), and observed under a light microscope (Olympus BX53).

Exposure experiment through drinking water using mice

Immature female CD-1 mice (20 days old) were obtained from Experimental Animal Tech Co. of Weitonglihua (Beijing, China). Mice with a maximal difference in body weight of 1 g were selected for the experiments and randomly assigned to either the treatment or control groups. The animals were housed four to a cage and acclimatized in a controlled environment with a temperature of 22 °C±2 °C, relative humidity between 40 and 60%, and artificial lighting in a 12 h/12 h light-dark cycle. The animals were fed ad libitum an oestrogen-free diet from Trophic Animal Feed High-tech Co., Ltd. (Nantong, China), and drinking water was provided ad libitum in glass bottles. Before the experiments, the mice were randomly assigned to five groups (n=8). Ultra-pure water was used as a control. FULV was dissolved in ultra-pure water to a concentration of 100 ng l−1 and used as a positive control. BHPF was dissolved in ultra-pure water to prepare a concentration of 100 ng l−1, which is relevant to human exposure. Two plastic drinking bottles labelled ‘BPA-free’, of different brands, were purchased and denoted Bottle A and Bottle B. The plastic bottles were filled with boiling ultra-pure water from a stainless steel electric water heater and allowed to cool down to room temperature before the animal experiment. The BHPF levels in the cooled boiled waters from Bottle A, Bottle B and the stainless steel electric water heater were determined by GC–MS. No BHPF was detected in the cooled boiled water from the stainless steel electric water heater. The cooled boiled waters from Bottle A and Bottle B were transferred to glass bottles during the experiment. The exposure experiment was performed for 10 days beginning on PND 24. After the period of exposure, the mice were weighed and killed in turns for each group, whereby in each turn only one mouse in a group was killed. Blood was collected by cardiac puncture soon after each animal was killed, and the uteri were removed, blotted and weighed by one experimenter to reduce the risk of bias in the data collection and immediately frozen in liquid nitrogen for gene expression analysis. The relative uterine weight was calculated to evaluate the anti-uterotrophic activity. The serum was separated by centrifugation and frozen at −20 °C for BHPF analysis. The serum BHPF levels were analysed by GC–MS using the same method as that for human serum. The gene expressions of sprr2a and sprr2b in the uteri were determined by Q-RT-PCR.

Data analysis

Data analysis was performed with the statistical program SPSS (v.18.0; Chicago, IL). The data are presented as the mean and s.d. unless otherwise indicated. Group differences were assessed by one-way analysis of variance and Fisher’s LSD tests after running the one-sample Kolmogorov–Smirnov test and the test of homogeneity of variances. P values of <0.05 were considered statistically significant.

Data availability

Microarray data are deposited in Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) under accession code GSE74066. All the other data supporting the findings of this study are available from the corresponding authors on request.