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Ecotoxicology and Environmental Safety
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ECOTOXICOLOGY ENVIRONMENTAL SAFETY
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Fluorene-9-bisphenol regulates steroidogenic hormone synthesis in H295R cells through the AC/cAMP/PKA signaling pathway
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Yuan Huang ª, Wei Zhangª,”, Na Cuiª, Zhiming Xiaoª, Wenyu Zhaoª, Ruiguo Wangª, John P. Giesy b,c,d, Xiaoou Sua,’
a Institute of Quality Standards and Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Beijing 10081, China
b Department of Veterinary Biomedical Sciences, and Toxicology Center, University of Saskatchewan, 52 Campus Dr, Saskatoon, SK S7N 5B4, Canada
” Department of Integrative Biology and Center for Integrative Toxicology, Michigan State University, 784 Wilson Rd, East Lansing, MI 48824, USA d Department of Environmental Science, Baylor University, 97266 One Bear Place, Waco, TX 76798, USA
ARTICLE INFO
Edited by Professor Bing Yan
Keywords:
BHPF Steroidogenesis Endocrine disruption Signal transduction In vitro
ABSTRACT
Fluorene-9-bisphenol (BHPF), which has been used as a substitute for bisphenol A (BPA) in consumer goods and industrial products, can be detected in environmental media and human urine. BHPF has been reported to have endocrine-disrupting effects, whereas deleterious effects on steroidogenesis in H295R cells and underlying mechanisms are still unclear. Here, we investigated effects of BHPF on steroidogenesis using human adreno- cortical carcinoma cells (H295R). Cytotoxicity was initially assessed and half-maximal inhibitory concentration (IC50) was determined based on proliferation of cells. Responses of four steroid hormones, aldosterone, cortisol, testosterone and 17ß-estradiol (E2), and ten critical genes, StAR, HMGR, CYP11A1, CYP11B1, CYP11B1, HSD3B2, CYP21, CYP17, 17ß-HSD, and CYP19, involved in steroidogenesis after exposure to non-cytotoxic concentrations of BHPF were determined in the presence or absence of 100 uM dbcAMP. Adenylate cyclase (AC) activity, intracellular concentrations of cAMP, PKA activity and amounts of steroidogenic factor-1 (SF-1) gene and ex- pressions of proteins were determined to elucidate underlying mechanisms of effects on steroidogenesis. BHPF was cytotoxic to H295R cells in a dose- and time-dependent manner. Effects on production of hormones results demonstrated that exposure to greater concentrations of BHPF inhibited productions of aldosterone, cortisol, testosterone and E2 by down-regulation of steroidogenic genes. Inhibition of AC activity, intercellular cAMP content and PKA activity after exposure to BHPF implied that the AC/cAMP/PKA signaling pathway was involved in BHPF-induced suppression of steroidogenesis in H295R cells. Additionally, BHPF inhibited ste- roidogenesis and expressions of steroidogenic genes via decreasing expression of SF-1 protein, both in basal and dbcAMP-induced treatment. These results contributed to understanding molecular mechanisms of BHPF-induced effects on steroidogenesis and advancing the comprehensive risk assessment of BPs.
1. Introduction
Bisphenols are widely produced for the production of polycarbonate (PC), epoxy resins, and polyurethane, which are used in numerous daily necessities, including paper cups, can coatings, plastic products, thermal paper, and electronics (Karrer et al., 2019; Xue et al., 2019). Humans are frequently exposed to bisphenols (BPs) in daily life, which have adverse effects on human health, especially the endocrine-disrupting effects.
Fluorene-9-bisphenol (BHPF) is one of the alternatives to bisphenol- A (BPA) and can also be used to produce polymer materials (Liu et al.,
2008; Zhen et al., 2009), which are applied to produce protective coatings, insulation materials, and adhesives as well as daily necessities such as bottles for drinking water, takeaway packaging, and coatings of metal cans. BHPF can be detected in natural water sources (Jin and Zhu, 2016). BHPF has been found in imported PC plastic bottles and their bottled water and BPA-free bottled water (Zhang et al., 2017). BHPF is also detected in the blood serum of college students with a mean con- centration of 0.34 ± 0.21 ng/ml (Zhang et al., 2017), which is greater than that of the substitutes for BPA, bisphenol-F (BPF) and bisphenol-S (BPS), which have mean concentrations of 0.087 ng/ml and 0.0089
* Corresponding authors. E-mail addresses: Huangyuan_171@126.com (Y. Huang), zhangwei03@caas.cn (W. Zhang), cuina0423@163.com (N. Cui), xiaozhiming@caas.cn (Z. Xiao), 17864272808@163.com (W. Zhao), wangruiguo@caas.cn (R. Wang), jgiesy@aol.com (J.P. Giesy), suxiaoou@caas.cn (X. Su).
ng/mL, respectively, in blood serum of people living near e-waste sta- tions (Song et al., 2019).
Recently, results of studies have demonstrated potential hazards of BHPF to the health of animals or humans. BHPF has been reported to be an anti-estrogen, which caused reduced mass of the uterus, induced endometrial atrophy and adversely affected pregnancy in mice (Zhang et al., 2017). In the reporter gene bioassays for detecting activities of BHPF against steroid hormone receptors, BHPF was found to be a potent antagonist for ER but not for androgen (AR), progesterone (PR), gluco- corticoid (GR) or mineralocorticoid (MR) (Grimaldi et al., 2019). IC50 values based on the antagonism of BHPF as determined using the dual-luciferase report test, to ERa and ERß are 109 nM and 75.3 nM, respectively (Zhang et al., 2017). These results further indicate that BHPF can exert anti-estrogen effects via competitively binding to the ER. BHPF not only interferes with the endocrine system but also has adverse effects on reproduction and potential behaviors (Mi et al., 2019). BHPF has toxic effects on the ovaries of different species due to inhibition of maturation and development of oocytes, and reduced quality of oocytes due to damage to the cytoskeleton, oxidative stress, damage of DNA, mitochondrial dysfunction, and cell apoptosis (Jia et al., 2019; Jiao et al., 2019, 2020). BHPF can interfere with male and female zebrafish’s courtship preferences, induce anxiety and depression-like symptoms, and down-regulate the expression of estrogen receptors (esr1 and esr2a) and steroid hormone synthesis-related genes (hsd3b, cyp17a1 and cyp19a1a) (Mi et al., 2019).
Steroid hormones are a class of tetracyclic aliphatic, hydrocarbon compounds produced in steroid-synthesizing cells in adrenal glands, ovaries, testes, and brain. The adrenal gland, which is responsible for the production of three major types of steroid hormones: mineralocorti- coids, glucocorticoids, and sex hormones is one of the most important endocrine organs for maintaining homeostasis in humans (Li et al., 2013). It has been reported that the effects of BPA, BPS, BPF, and BPAF on the steroidogenesis and steroidogenic gene expressions in H295R cells (Zhang et al., 2011; Feng et al., 2016), but the effects of BHPF on steroidogenesis are still unknown. BHPF has been found to down-regulate some steroidogenic gene expression on zebrafishes (Mi et al., 2019), mechanisms involved in the mediation of steroidogenesis are still lacking.
To elucidate the effects of BHPF and underlying mechanisms of ef- fects of BHPF on steroidogenesis, human adrenocortical adenocarci- noma cell line (H295R) was used to measure secretion levels of steroid hormones as well as expressions of enzymes and genes in the steroido- genic signaling pathway. H295R cell line is currently an ideal model for studying steroid hormone synthesis since it retains all steroidogenic pathways and secretes all steroid intermediates of steroidogenesis (Gazda et al., 1990; Hecker et al., 2006; Hecker and Giesy, 2008). In this study. Cytotoxicity and effects on the secretion of steroid hormones were analyzed in H295R cells after exposure to BHPF, and the role of the AC/cAMP/PKA signaling pathway in modulating adverse effects of BHPF was investigated.
2. Materials and methods
2.1. Chemicals
Fluorene-9-bisphenol (BHPF, CAS no. 3236-71-3, 97.5% purity) was purchased from Dr. Ehrenstorfer Inc. (Augsburg, Germany). Dibutyryl- CAMP sodium salt (dbcAmp, CAS no. 16980-89-5, 99.71% purity) and forskolin (FSK, CAS no. 66575-29-9, 99.82% purity) were obtained from MedChemExpress Co. Ltd. (Monmouth Junction, USA). Dimethyl sulfoxide (DMSO) was purchased from Sigma (St.Louis, USA). All drugs were dissolved in DMSO to form the stock solutions.
2.2. Cell culture
H295R cell line was obtained from Cell Bank (China Academy of
Medical Sciences, Institute of Basic Medical Center of Cell). Cells were cultured in 75-cm2 flasks containing Dulbecco’s Modified Eagle Medium (DMEM)/F-12 (Gibco, Grand Island, USA) supplemented with 1% insulin-transferrin-selenium-G (ITS-G, Gibco, Grand Island, USA), 5.35 ug/mL linoleic acid (Sigma, USA), 1.25 mg/mL bovine serum albumin (BSA, Sigma, USA), 1% penicillin-streptomycin (Gibco, USA) and 2% Ultroser G (Pall, Port Washington, USA) at 37 ℃ in a humidified at- mosphere containing 5% CO2. The exposure medium consisted of phenol red-free DMEM/F12 medium supplemented with 1% ITS-G, 1% penicillin-streptomycin, and 10% charcoal-stripped FBS. BHPF dis- solved in DMSO were added to the medium after cells adhered for 24 h. The final concentration of DMSO in the culture medium was ≤ 0.1% (v/ v). Control cells were incubated with 0.1% DMSO to match the final concentration achieved in culture medium in the experimental exposures.
2.3. Cell viability assay
In the cell viability assay, H295R cells were seeded into 96-well microplates with a density of 3 × 104 cells/well. Cell counting kit-8 (CCK-8, Dojindo, Japan) assay was utilized to detect the cell viability of H295R cells exposed to BHPF. Cells were treated with BHPF (0, 10, 20, 30, 40, 50, 60, 70 or 100 µM) alone for 24 h, 48 h and 72 h, or combined with 100 uM dbcAMP for 48 h. For each duration of exposure, the CCK-8 reagent was added to the culture medium and incubated for 3 h at 37 ℃. Absorbance was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA), and the 650 nm was set as the reference wavelength to deduct the background interference caused by the turbid media. The assay was conducted in triplicate with six repli- cates of each concentration. The relative cell viability was presented as a percentage of treatment groups compared to the DMSO control group, which are subtracted from the absorbance of the background control.
2.4. Cell proliferation assay
H295R cells were grown in 96-well microplates with a density of 2 x 104 cells/well for 24 h to adhere. Proliferation of H295R cells exposed to 0, 10, 20, 30, 40 and 50 µM BHPF for 48 h was assessed by a BeyoClick EdU-555 cell proliferation assay kit (Beyotime, Shanghai, China). Ac- cording to the instruction of the kit, 10 mM EdU was diluted to 5 AM from fresh cell medium to prepare the EdU working solution. The BHPF- containing cell culture medium was removed and the fresh EdU working solution was added in 100 µL/well and incubated at 37 ℃ in the dark for 2 h. H295R cells were fixed with 4% paraformaldehyde for 15 min, washed three times and permeabilized with 0.3% Triton X-100 for 15 min. H295R cells were washed twice and incubated with click reaction buffer in the dark for 30 min at room temperature. Finally, cells were counterstained with 1 x Hoechst 33342 (10 µM) and incubated in the dark for 10 min. After being washed three times, cells were observed and the images were analyzed with a high-content screening system (Per- kinElmer, USA).
2.5. Hormone measurement
H295R cells were cultured in DMEM/F-12 without phenol-red and seeded into 24-well plates at a density of 2.5 x 105 cells/well. After 24 h of pre-culture to adhere completely, cells were exposed to 0, 0.01, 0.1, 1, 10, 20 or 30 µM BHPF in the basal treatment or 100 M dbcAMP- induced treatment, respectively. The DMSO group was the control of the basal treatment, and the dbcAMP-induced group was the control of the dbcAMP-induced treatment. After exposure for 48 h, cell culture medium in each well was collected and stored at - 20 ℃ for hormone measurement. Concentrations of aldosterone, cortisol, testosterone (T) and 17ß-estradiol (E2) were determined, using commercially available radioimmunoassay kits from Beijing North Institute of Biological Tech- nology according to the manufacturer’s instructions. Sensitivities of
each RIA kit was 0.02 ng/mL (for aldosterone), 2 ng/ml (for cortisol), 0.02 ng/ml (for T) and 5 pg/mL (for E2), respectively. The intra-assay and inter-assay coefficients of variation were less than 10% and 15%, respectively.
2.6. RNA extraction and RT-qPCR
H295R cells were grown in 6-well cell culture plates with a density of 1 x 106 cells/well. When the cell confluence reached approximately 70%, H295R cells were exposed to 0, 0.01, 0.1, 1, 10, 20 and 30 µM BHPF in the presence or absence of 100 µM dbcAMP for 48 h. Total RNA was isolated from the cells by use of the RNAprep Pure Cell Kit (TIAN- GEN, Beijing, China) following the manufacturer’s instruction. Con- centrations of total RNA were measured at 260 nm using NanoDrop (Thermo Scientific, USA). All RNA samples exerted suitable A260/A280 and 28 S/18 S ratios. Reverse transcription of RNA and PCR amplifica- tion was performed using the One Step TB Green PrimeScript RT-PCR Kit (Takara, Japan) with QS5 real-time PCR system (Applied Biosystem Inc., USA). PCR primers were listed in Table S1. Target gene (StAR, HMGCR, CYP11A1, CYP11B1, CYP11B1, CYP19, CYP17, CYP21, 17ß-HSD, HSD3B2, SF-1) expression levels were normalized to ß-actin expression values and calculated using the 2-AACt method (Livak and Schmittgen, 2001). The amplification efficiencies of primers varied in the range of 93.3%- 104.2%, which has met the criteria for high amplification ef- ficiency of RT-qPCR.
2.7. Quantification of AC activity and intracellular cAMP
H295R cells were seeded into T25 cell culture flask at a density of 3 x 106 cells/well and cultured for 24 h. After the exposure of 30 µM BHPF or in the presence of 10 µM FSK to H295R cells for 48 h, the adenylate cyclase (AC) activity was determined by the amount of cAMP produced. Isolation of cell membrane proteins was performed using the CelLytic MEM Protein Extraction Kit (Sigma, USA) by two steps according to the manufacturer’s instruction, including cell collection and separation of hydrophobic and hydrophilic proteins. The lower hydrophobic phase enriched with hydrophobic and raft associated proteins was retained for measurement of AC activity according to the method described previ- ously (Wang et al., 2015). Membrane protein (30 µg) was added to the reaction buffer (50 mM Tris-HCl, pH 7.4; 5.0 mM MgCl2; 0.5 mM EDTA; 1 mM ATP; 0.1 mM GTP; 0.2 IU pyruvate kinase; 0.1 unit of myokinase and 2.5 mM phosphoenolpyruvate), and incubated at 37 ℃ for 15 min. The converted cAMP content was detected by enzyme-linked immuno- sorbent assay (ELISA) kit (Cayman, Michigan, USA).
Intracellular concentrations of cAMP were determined by use of the CAMP ELISA kit based on the manufacturer’s instructions. Absorbance was measured at 410 nm by a microplate reader, and cAMP content was calculated in accordance with the standard curve.
2.8. PKA activity assay
H295R cells were exposed for 48 h to 30 µM BHPF in the presence or absence of 100 µM dbcAMP or 10 µM FSK. Cell lysates were obtained by sonication in an ice bath, and PKA activity was analyzed using ELISA kits (Invitrogen, Frederick, USA) according to the manufacturer’s instruc- tion. In brief, the appropriate samples and reconstituted adenosine triphosphate (ATP) were added into each well of the microtiter plate and incubated for 90 min at 30 ℃ with shaking. Then, the detection antibody was added and incubated for 60 min at room temperature. The chro- mogen was finally added and incubated for 30 min at room temperature. After adding the stop solution, the absorbance was measured at 450 nm.
2.9. Western blotting
Total protein was extracted from cells using RIPA lysis buffer (Beyotime, Shanghai, China) with a protease inhibitor PMSF and a
phosphatase inhibitor (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Concentrations of protein were determined by a BCA Protein Assay Kit (TIANGEN, Beijing, China). Protein samples (30 µg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electro-blotted onto a polyvinylidene difluoride (PVDF) membrane. Subsequently, membrane was blocked in 5% skimmed milk and incubated overnight at 4 ℃ with primary anti- bodies against phospho-PKA substrate (#9624 S, CST, USA), SF-1 (#12800 S, CST, USA) and ß-actin (#4970 S, CST, USA). After washing three times with TBST, the PVDF membrane was incubated with the anti-rabbit IgG-HRP antibody (#7074 S, CST, USA) for 1 h at room temperature. Immunoblots were prepared with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo, USA), and detected by Tanon 5200 chemiluminescence/fluorescence image analysis system (Tanon, Shanghai, Beijing). The binds intensity was quantified using ImageJ software (LiCor, Lincoln, NE, USA). Relative expression of each protein was calculated by normalization to ß-actin, and the resulting ratios in the control group were normalized to 1.
2.10. Statistical analysis
The analysis of hormone production and gene transcription profiles was processed with SPSS 25.0 for windows (SPSS Inc., Chicago, USA). A Shapiro-Wilks normality test indicated that parameters met the assumption of normal distribution required for the application of para- metric statistical inferences. The assumption of homogeneity of variance was assessed using Levene’s test. The statistical significance of differ- ences was calculated using one-way analysis of variance (ANOVA) fol- lowed by post hoc LSD tests. A value of p < 0.05 was considered statistically significant. The IC50 values of the cytotoxic of BHPF and all result graphs were performed using GraphPad Prism 9.0 (GraphPad Software Inc. San Diego, USA). The results were represented as mean with standard deviation (SD) in triplicate within three independent experiments.
3. Results
3.1. Effects of BHPF on cell viability and proliferation
The IC50 values for H295R cells exposed to BHPF for 24 h, 48 h, 72 h were 48.82 uM, 44.28 µM and 40.28 uM, respectively. These results indicated that BHPF was cytotoxic to H295R cells in a dose- and time- dependent manner (Fig. 1 A). Meanwhile, no significant cytotoxicity on H295R cells of BHPF were observed at concentrations of 0.01, 0.1, 1, 10, 20 and 30 µM in the presence of 100 µM dbcAMP (Fig. 1B). However, significant cytotoxicity was observed in cells exposed to 40 µM BHPF(P < 0.01). In addition, 40 µM BHPF significantly inhibited DNA synthesis of H295R cells in the BeyoClick EdU-555 cell proliferation assay (P < 0.01), exerting significant cytotoxicity on the cell proliferation (Fig. 1 C and Fig. 1D). Thus, concentrations of 0.01, 0.1, 1, 10, 20 and 30 µM BHPF were selected for further research.
3.2. Effects of BHPF on the production of steroid hormones
Effects of BHPF (0.01-30 µM) on the steroid hormones produced by H295R cells were investigated in the presence or absence of dbcAMP (Fig. 2). Compared to the DMSO control, levels of aldosterone and cortisol were not significantly altered at tested concentrations ranging from 0.01 to 10 µM. However, aldosterone production decreased to 77% and 36%, and cortisol production decreased to 71% and 35% after 20 µM and 30 µM BHPF exposure, respectively (Fig. 2 A and Fig. 2B). In dbcAMP-induced exposure, the production of aldosterone and cortisol was significantly increased (P < 0.01), whereas they were inhibited after exposure to 20 µM or 30 µM BHPF. Compared to the dbcAMP control, aldosterone production was reduced to 76% and 78%, and cortisol production was reduced to 53% and 14% (Fig. 2 A and Fig. 2B).
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Fig. 1. Cytotoxicity of BHPF on H295R cells. (A) Cell viability of H295R cells exposed to 0-100 µM BHPF for 24 h (orange), 48 h (black), and 72 h (blue); (B) Cell viability of H295R cells exposed to 0-40 AM BHPF in the presence of 100 µM dbcAMP for 48 h; (C) Micrographs presenting the incorporation of EdU in H295R cells exposed to BHPF or combined with 100 µM dbcAMP for 48 h. Red: the EdU positive cells; blue: the cell nuclei (Hoechst); (D) Cell proliferation level was quanti- tatively analyzed by the percentage of EdU positive cells versus total cell numbers. Results are means ± SD of three independent experiments conducted in 6 wells per exposure dose. Compared to the DMSO control, * P < 0.05, * * P < 0.01; Compared to the dbcAMP-induced control, P < 0.05, P < 0.01.
No statistically significant changes were observed for aldosterone and cortisol production in cells exposed to 0.01-10 µM BHPF (Fig. 2 A and Fig. 2B).
Compared to the DMSO control cells, the production of testosterone increased to 1.40-fold in cells exposed to 0.01 µM BHPF, whereas it declined at greater concentrations of BHPF in a dose-dependent manner (Fig. 2 C). A significant reduction in testosterone production was observed at 10, 20 and 30 µM BHPF to 31%, 31% and 14%, respectively (Fig. 2 C). Exposure to 0.01 µM BHPF increased testosterone production to 1.24-fold relative to the dbcAMP-induced control, while testosterone production was reduced to 74% in cells exposed to 30 µM BHPF (Fig. 2 C). The production of E2 was 1.33-fold greater in cells exposed to 1 µM BHPF than that of the DMSO control but decreased to 38% in cells exposed to 30 µM BHPF (Fig. 2D). In the presence of dbcAMP, no sig- nificant effects on E2 production were observed in cells exposed to concentrations of BHPF from 0.01 to 20 µM (Fig. 2D). Nevertheless, the production of E2 significantly decreased to 57% in cells exposed to 30 µM BHPF (Fig. 2D).
3.3. Effects of BHPF on the transcription of steroidogenic genes
Transcriptions of critical genes encoding enzymes involved in ste- roidogenesis, including HMGR, StAR, CYP11A1, CYP11B1, CYP11B1, CYP19, CYP17, CYP21, 17ß-HSD and HSD3B2, were affected by expo- sure to BHPF (Fig. 3). Compared to the DMSO control, expression of HMGR was down-regulated to 83%, 87%, 86% and 76% in cells exposed to concentrations of BHPF from 1 to 30 µM, and to 82% and 62% of the dbcAMP-induced control in cells exposed to 20 µM or 30 µM BHPF
(Fig. 3A). Expression of StAR was down-regulated to 87%, 82% and 66% relative to the DMSO control in cells exposed to 1, 20 and 30 µM BHPF, while StAR was downregulated to 66% of the dbcAMP-induced control when cells were exposed to 30 µM BHPF (Fig. 3B). Expression of CYP11A1 was down-regulated to 78-89% of the DMSO control in cells exposed to 0.1-30 µM BHPF, and to 80% and 66% of the dbcAMP- induced control when exposed to 20 µM and 30 µM BHPF, respec- tively (Fig. 3C). Expression of HSD3B2 was downregulated to 80% and 42% of the DMSO control, and 72% and 45% of the dbcAMP-induced control when cells were exposed to 20 µM and 30 µM BHPF, respec- tively (Fig. 3D).
Expression of CYP21A2 was not significantly altered by exposure to 0.1-30 µM BHPF, whereas exposure to 1-20 µM BHPF down-regulated expression of CYP21A2 to 73-83% relative to the dbcAMP-induced control (Fig. 3E). Expression of CYP11B1 was significantly down- regulated to 72% and 41% of the DMSO and dbcAMP-induced control in cells exposed to 30 µM BHPF (Fig. 3F). Exposure to 30 µM BHPF also down-regulated CYP11B2 to 25% and 36% of the DMSO and dbcAMP- induced control (Fig. 3G). When H295R cells were exposed to 30 µM BHPF, CYP17A1 was down-regulated to 73% and 63% of the DMSO and dbcAMP-induced control (Fig. 3H). In the basal treatment, 0.1 µM BHPF up-regulated 17/HSD to 1.12-fold, while 20 µM and 30 µM BHPF down- regulated 17ßHSD to 82% and 84%. In dbcAMP-induced cells, concen- trations in the range of 0.01-1 µM BHPF significantly up-regulated expression of 17HSD to 1.25-fold, 1.14-fold and 1.15-fold (Fig. 3I). Exposure to 0.01-30 µM BHPF down-regulated CYP19A1 to 49-80% of the DMSO control and 44-87% of the dbcAMP control in a dose- dependent manner (Fig. 3J).
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3.4. Effects of BHPF on AC activity and intracellular cAMP content
FSK is used to increase the level of intracellular cAMP via directly activating AC. The second-messenger cAMP plays a pivotal role in regulating steroidogenesis and other diverse cell functions. To further investigate the mechanism of inhibition on steroidogenesis of BHPF, we analyzed intracellular cAMP content and AC activity in H295R cells treated with BHPF alone or combined with FSK. As is shown in Fig. 4, 10 µM FSK increased AC activity and the level of intracellular cAMP to 1.56-fold and 2.86-fold of the DMSO control. When H295R cells were exposed to 30 µM BHPF, a declining trend of AC activity and cAMP content was observed, but no statistical differences compared to the DMSO control group. However, BHPF significantly inhibited AC activity to 38.4% and decreased cAMP content to 71.9% in the presence of FSK, compared to the FSK-induced control.
3.5. Effects of BHPF on PKA activity
One of the most important target proteins in the downstream signaling pathway of cAMP is PKA. The various physiological functions of cAMP are to activate PKA to phosphorylate its substrates, thereby regulating the expression of various key downstream factors. In order to further clarify the regulatory role of the cAMP/PKA signaling pathway in the inhibition of steroid hormone synthesis by BHPF, the effects of BHPF in the basal, FSK -induced and dbcAMP-induced treatments on PKA activity were examined by use of ELISA, and the phosphorylation substrate of PKA was analyzed by western blotting. Both 100 µM
dbcAMP and 10 µ M FSK can activate PKA to 1.45-fold and 1.57-fold of the DMSO control, whereas PKA activity was suppressed to 76%, 78% and 67% of the corresponding control after exposure to 30 µM BHPF in the basal, dbcAMP-induced or FSK-induced treatments (Fig. 5).
3.6. Effects of BHPF on SF-1 expression
SF-1 is a critical regulator in steroid hormone synthesis. In order to further confirm the involvement of SF-1 in the BHPF-suppressed ste- roidogenesis, the alteration of gene and protein expressions of SF-1 was investigated (Fig. 6). In the basal treatment, 30 uM BHPF up-regulated expression of SF-1 to 1.70-fold relative to the DMSO control, whereas down-regulated the protein expression of SF-1-62%. In the dbcAMP- induced treatment, dbcAMP significantly up-regulated the gene and protein expressions of SF-1-2.12-fold and 1.69-fold of the DMSO con- trol, while 30 µM BHPF inhibited both gene and protein expression of SF-1-78% and 66% of the dbcAMP-induced control.
4. Discussion
The dose- and time-dependent cytotoxicity of BHPF to H295R cells observed in this study was consistent with similar effects of BPA, BPF, BPS, and BPAF, all of which have been reported to cause cytotoxicity to H295R cells (Feng et al., 2016). Based on the lesser IC50 for BHPF, it was more potent than BPA and its other analogues. The response concen- tration of BHPF might vary in different cell types. After human endo- metrial cancer Ishikawa cells were exposed to 50 µM BHPF for 24 h, the
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0.0
0
0.01
0.1
1
10
20
30
Concentration of BHPF(UM)
(E)
(F)
Concentration of BHPF(UM)
Relative mRNA expression level
5.0-
CYP21A2
Basal
Relative mRNA expression level
120
CYP11B1
Basal
dbcAMP
dbcAMP
90
3.5.
#
60
2.0
N
30
₿
1.5
1.5
1.0-
1.0
**
0.5
0.5
0.0
0.0
0
0.01
0.1
1
10
20
30
0
0.01
0.1
1
10
20
30
Concentration of BHPF(UM)
Concentration of BHPF(UM)
(G)
Relative mRNA expression level
28
CYP11B2
Basal
Relative mRNA expression level
(H)
CYP17A1
Basal
dbcAMP
8
6
dbcAMP
23
#
18
6
#
13
4
8
#
3
2
1.5
1.5
1.0.
1.0.
**
**
**
0.5.
**
0.5
0.0
0.0
0
0.01
0.1
1
10
20
30
0
0.01
0.1
1
10
20
30
Concentration of BHPF(UM)
Concentration of BHPF(UM)
Relative mRNA expression level
(1)
Relative mRNA expression level
(J)
17ßHSD
CYP19A1
1.5
Basal
dbcAMP
30
Basal
25.
dbcAMP
#
*
20
#
1.0.
T
T.
15-
**
*
#
10
5
1.5
0.5
1.0
**
**
**
**
**
0.5
**
0.0
0.0
0
0.01
0.1
1
10
20
30
0
0.01
0.1
1
10
20
30
Concentration of BHPF(UM)
Concentration of BHPF(M)
(A)
(B)
600
10
**
AA
AC activity(pmol/min/mg)
*
AA
CAMP content (pmol/mL)
8
400
6
200
4
2
0
0
FSK
-
-
+
+
FSK
-
-
+
+
BHPF
+
+
BHPF
-
-
-
+
-
+
(A)
(B)
3.
**
245 kDa
135 kDa
PKA activity (U/mg protein)
2.
100 kDa 80 kDa
*
Phospho-PKA substrates
1-
46 kDa
32 kDa
0
T
T
dbcAMP
ß-actin
-
-
+
+
-
-
FSK
-
-
-
-
+
+
BHPF
-
BHPF
+
-
+
-
+
-
+
-
+
-
+
dbcAMP
-
-
+
+
-
-
FSK
-
-
-
-
+
+
cell viability was reduced to about 50%. However, no significant change in cell viability was observed with concentrations of BHPF increasing to 100 µM (Wang et al., 2019). In our work, the IC50 value (48.8 µM) of BHPF at 24 h was at the level comparable with the above, but the cell viability of H295R cells was significantly decreased to 8.3% and 7.6% of the control group after 60 UM and 100 µM BHPF exposure. In contrast, no significant effect on cell viability of the porcine Sertoli cells and meiosis in mouse oocytes were found after exposure to 50 µM BHPF for 24 h (Jiao et al., 2019; Zhang et al., 2021). Thus, our results indicated that H295R cells were sensitive to BHPF exposure. Since endocrine disrupting compounds (EDCs) can interfere with the endocrine system at concentrations that do not cause overt toxicity like inhibition of cell proliferation, the effects of BHPF on steroidogenesis and underlying mechanisms were investigated based on dosages of BHPF that had no remarkable cytotoxicity.
To the best of our knowledge, this is the first report of the adverse effects of BHPF in the basal and dbcAMP-induced treatments on ste- roidogenesis in H295R cells. The dbcAMP, a derivative of cAMP, was used to mimic the secretion mode of adrenocorticotropic hormone (ACTH) (Feng et al., 2016) and activate the PKA signaling pathway to induce the secretion of progesterone and cortisol, as well as to increase the expression of critical genes involved in the pathway of steroid
hormone synthesis in H295R cells (Li et al., 2013). In our study, the significant inhibition of the production of aldosterone, cortisol, testos- terone and estradiol observed in H295R cells exposed to BHPF at greater concentrations in both basal and dbcAMP-induced treatments were similar to previous findings that BPA, BPS and BPAF could decrease the production of aldosterone and cortisol in H295R cells treated with dbcAMP (Feng et al., 2016). In addition, BPB was demonstrated to decrease the cortisol level in H295R cells (Rosenmai et al., 2014). Noticeably, the amount of testosterone produced was altered in a non-monotonic profile in H295R cells exposed to BHPF in the basal and dbcAMP-induced treatments. More testosterone was produced at lesser concentrations of BHPF, but less was produced at greater exposures of BHPF. However, BPA, BPS and BPAF were found to decrease testos- terone production in a dose-dependent manner in H295R cells (Gold- inger et al., 2015; Feng et al., 2016). Additionally, BHPF interfered with estradiol production in a non-monotonic profile similar to that with testosterone production in the basal treatment but different from the findings that BPA, BPF, BPB and BPE stimulated E2 production (Rose- nmai et al., 2014; Goldinger et al., 2015). The non-monotonic responses of BHPF on the production of testosterone and E2 probably indicated that complicated effects of BHPF on the sex hormone endocrine system, which is associated with reproductive functions. In addition, the effects
(A)
(B)
SF-1
2.5
**
#
SF-1
Relative SF-1 mRNA expression
2.0
**
T
1.5
ß-actin
1.0-
Relative SF-1 protein level (normalized to control group)
2.0
**
0.5
1.5-
0.0
T
T
T
T
*
dbcAMP
-
-
+
+
1.0-
BHPF
-
+
-
+
0.5-
0.0
T
T
T
T
dbcAMP
-
-
+
+
BHPF
-
+
-
+
of BHPF on steroidogenesis in H295R cells were partially different from those of BPA and other analogues, suggesting the need for study on endocrine disrupting effects for each BPs.
To elucidate the underlying mechanism of the effects on steroido- genesis of BHPF, transcriptions of 10 critical genes involved in ste- roidogenesis were analyzed. HMGR and StAR, the essential rate-limiting enzymes, play pivotal roles in cholesterol synthesis and the transfer from the outer mitochondrial membrane to the mitochondrial membrane, respectively (Yan et al., 2018). CYP11A1 and HSD3B2 catalyze choles- terol to produce progesterone, an essential precursor for other steroid hormones synthesis. Progesterone was catalyzed sequentially by CYP21A2, CYP11B1 and CYP11B2 to produce aldosterone (Nogueira and Rainey, 2010). And 17«-hydroxyprogesterone, which is converted from progesterone by CYP17A1, is catalyzed by CYP21A2 and CYP11B1 to produce cortisol (Wang et al., 2015). Hence, CYP11B1 and CYP11B2 are responsible for regulating the final rate-limiting of the synthesis of cortisol and aldosterone, respectively. In our research, 30 µM BHPF could inhibit the aldosterone and cortisol production through the down-regulation of HMGR, StAR, CYP11A1, HSD3B2, CYP11B1 and CYP11B2 in the basal and dbcAMP-induced treatments. Consistent with the findings of BHPF in our study, BPA, BPF and BPAF can block the synthesis of aldosterone and cortisol via down-regulating the tran- scription of the rate-limiting enzymes (Feng et al., 2016). However, the reduction in the production of aldosterone and cortisol was observed in 20 µM BHPF dbcAMP-induced group, concurrent with no significant alteration in expressions of StAR, CYP11B1 and CYP11B2. Similarly, exposure to BPS suppressed the production of aldosterone and cortisol with no changes in transcription of their rate-limiting enzymes (Feng et al., 2016). Greater concentrations of BHPF inhibited the production of aldosterone and cortisol, similar to other BPs, but through different mechanisms in the steroidogenic pathway.
CYP17A1 catalyzes the conversion of progesterone and 17x- hydroxyprogesterone to androstenedione, which is the precursor sub- stance from which testosterone and E2 are produced. 17ßHSD and CYP19A1 catalyze the rate-limiting reactions for the synthesis of testosterone and E2, respectively (Oskarsson et al., 2006). In the basal treatment, BHPF might inhibit testosterone production by down-regulation of CYP17A1 and 17ßHSD. In contrast, due to no sig- nificant alteration in expression of 17/HSD, the down-regulation of
CYP17A1 mainly reduced testosterone production in H295R cells exposed to BHPF in the dbcAMP-induced treatment. Similarly, exposure to BPA, BPF, BPS or BPAF decreased testosterone production by down-regulation of CYP17A1 rather than 17(HSD, which was the case in dbcAMP-induced H295R cells (Feng et al., 2016). Since expressions of CYP17A1 and 17HSD were not significantly altered after H295R cells were exposed to 10 µM BHPF in the basal treatment, a decrease in testosterone production occurred might be due to down-regulation of expression of other genes in the upstream steroidogenic pathway or changes in the translation of mRNA or post-modification of proteins. Results of this study indicated that the disrupting effect of BHPF on steroidogenesis for H295R cells was different in the presence and absence of dbcAMP and might be a function of exposure concentrations, as well as other confounders. Noticeably, lesser concentrations of BHPF might stimulate testosterone production in the basal and dbcAMP-induced cells through up-regulation of 17ßHSD. Also, down-regulation of expression of CYP19A1 might result in lesser con- version from testosterone to E2, thereby promoting testosterone secre- tion. The non-monotonic effect of BHPF on synthesis of testosterone was different from what has been observed for other BPs that only caused suppression (Zhang et al., 2011; Rosenmai et al., 2014; Goldinger et al., 2015). It suggested that more concerns about effects of BHPF on the androgen endocrine system and male reproduction are necessary, especially exposures to BHPF at low concentrations.
The down-regulated transcription of CYP19A1 was observed in a dose-dependent manner, whereas only BHPF at a concentration of 30 µM exhibited inhibition of E2 production. Assuming that the enzyme directly participates in regulation of steroidogenesis and down- regulated transcription levels of genes might be accompanied by un- changed or even increased amounts of protein. Thus down-regulation of CYP19A1 was not bound to decrease E2 production, especially accom- panying no remarkable alteration in upstream genes of the steroidogenic pathway. Exposure to BPF stimulated E2 production without changes in transcription of CYP19A1. Also, no significant alteration in expression of CYP19A1 was observed after exposure to BPA, BPS or BPAF (Feng et al., 2016). Compared to that finding, BHPF exerted a more sensitive effect on transcription of CYP19A1 than did other BPs. Thus, this might be used as a biomarker specific to the endocrine-disrupting effects of BHPF. Furthermore, down-regulation of CYP19A1 further demonstrated the
anti-estrogen activity of BHPF, which has been reported before (Zhang et al., 2017). Whether CYP19A1 could be a biomarker indicative of endocrine-disrupting effects deserves further investigation.
The cAMP/PKA signaling pathway plays an essential role in ste- roidogenesis. Activation of AC increases intracellular levels of the sec- ondary messenger cAMP, which ultimately activates the PKA signaling pathway. Steroidogenesis is mediated by the intracellular cAMP content,
which has been reported to stimulate production of steroid hormones (Yan et al., 2018; Jin et al., 2020). When activated PKA promotes phosphorylation of downstream target proteins and then recruits some co-activators, such as the cAMP response element-binding protein (CREB)-regulated transcription coactivators (CRTC) and SF-1 forming a complex to mediate steroidogenic acute regulatory protein (StAR) and expressions of cytochrome P450 1B1 (CYP1B1) expressions (Zheng and
BHPF
dbcAMP
Plasma Membrane
AC
Gas
GTP
(+)
Free cholesterol
MRAP
MC2R
B,Y
GDP
Gas
SF-1
BHPF
β,γ
CAMP
ATP
StAR
PKA
Steroid hormones
cholesterol
Mitochondria
30 µM BHPF exposure in the basal treatment
StAR
Cholesterol
CYP11A1
(Pregnenolone)
HSD3B2
HMGR
Progesterone
CYP21A2
(11- Deoxycorticosterone)
CYP11B1
(Corticoster one)
CYP11B2
Aldosterone
CYP17A1
CYP17A1
(17a-OH- Pregnenolone)
HSD3B2
(17a-OH- Progesterone)
CYP21A2
(11-Deoxycortisol)
CYP11B1
Cortisol
CYP17A1
CYP17A1
(DEHA)
HSD3B2
(Androstenedio ne)
17₿HSD
Testosterone
Significant decreased in hormone levels
CYP19A1
CYP19A1
Significant down-regulation in gene expression levels
(Estrone)
17₿HSD
17₿-Estradiol
No significant change
30 AM BHPF exposure in the dbcAMP-induced treatment
StAR
Cholesterol
CYP11A1
(Pregnenolone)
HSD3B2
Progesterone
CYP21A2
(11- Deoxycorticosterone)
CYP11B1
(Corticoster one)
CYP11B2
HMGR
Aldosterone
CYP17A1
CYP17A1
(17a-OH- Pregnenolone)
HSD3B2
(17a-OH- Progesterone)
CYP21A2
(11-Deoxycortisol)
CYP11B1
Cortisol
CYP17A1
CYP17A1
(DEHA)
HSD3B2
(Androstenedio ne)
17₿HSD
Testosterone
CYP19A1
CYP19A1
(Estrone)
17₿HSD
17B-Estradiol
Jefcoate, 2005; Smith et al., 2020). In the present study, BHPF signifi- cantly reduced concentrations of intracellular cAMP via attenuating the AC activity induced by FSK, an activator of AC activity. The decreased cAMP content led to inhibition of PKA activation in both BHPF dbcAMP-induced and FSK-induced cells. However, only significant in- hibition of PKA activity was observed in the BHPF basal treatment, indicating that distinct profile of the AC/cAMP/PKA signaling pathway was involved between the basal and dbcAMP-induced treatments. Noticeably, PKA might play a pivotal role in BHPF-induced suppression of steroidogenesis. Alteration of PKA activity contributed to BPA-induced dysfunction of spermatozoa in adult rats (Wan et al., 2016) or F1 adult mice (Rahman et al., 2017). Additionally, further study on its downstream signal molecules, such as CREB, CRTC, and SF-1, is essen- tial due to their important regulation in the transcription of steroido- genic genes. A recent report demonstrated that BPA has sex-specific effects on learning and memory, which is associated with down-regulation of PKC/ERK/CREB signaling pathway (Wu et al., 2020).
Previous reports have demonstrated that SF-1, a key steroidogenic transcription factor, plays a pivotal role in steroidogenesis by regulating the transcription of steroidogenic genes, including StAR, CYP11A1, CYP1B1, and CYP19A1 (Meinsohn et al., 2019; Liu et al., 2020). Although it has been found that BPA could not alter adrenal expression of SF-1 in adult mouse offspring (Medwid et al., 2016), our results showed that decreased expression of SF-1 protein after BHPF exposure was directly involved in down-regulation of critical steroidogenic genes in both the basal and dbcAMP-induced treatments. Interestingly, BHPF could stimulate the transcription of SF-1 while inhibiting the protein expression of SF-1 in the basal treatment. It suggested that BHPF might be inclined to inhibit translation of SF-1 and even post-translation modification, ultimately leading to reduced protein expression of SF-1. The inconsistency of changes in SF-1 gene and protein expression after BHPF basal exposure might be associated with post-transcriptional regulation, indicating that more complicated mechanisms may be involved in the effects of BHPF on steroidogenesis.
5. Conclusions
In conclusion, our findings firstly demonstrated that BHPF could alter steroidogenesis in H295R cells at concentrations that did not alter proliferation. Relative in the basal and dbcAMP-induced cells, ste- roidogenesis was decreased due to down-regulation of steroidogenic genes in cells exposed to greater concentrations of BHPF. Blockage of the AC/cAMP/PKA pathway and down-regulation of SF-1 were involved in BHPF-suppressed steroidogenic gene expressions and steroidogenesis (Fig. 7). Further in vitro and in vivo studies are needed to elucidate the mechanisms of the endocrine disruption effects and assess the potential health risks of BHPF, especially BHPF exposure at low concentrations.
CRediT authorship contribution statement
Yuan Huang: Conceptualization, Methodology, Formal analysis, Data curation, Visualization, Investigation, Writing - original draft, Writing - review & editing; Wei Zhang: Writing - review & editing, Conceptualization, Validation; Na Cui: Data curation, Validation; Zhiming Xiao: Conceptualization, Resources; Wenyu Zhao: Investiga- tion; Ruiguo Wang: Resources; John P. Giesy: Writing - review & editing; Xiaoou Su: Project administration & supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
The study was financially supported by the National Key Reasearch and Development Program of China (No.2017YFC1600303) and the Innovation Program of the Chinese Academy of Agricultural Science (Feed Quality and Safety).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2022.113982.
References
Feng, Y., Jiao, Z., Shi, J., Li, M., Guo, Q., Shao, B., 2016. Effects of bisphenol analogues on steroidogenic gene expression and hormone synthesis in H295R cells. Chemosphere 147, 9-19.
Gazda, A.F., Oie, H.K., Shackleton, C.H., Chen, T.R., 1990. Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis. Cancer Res 50, 5488-5496.
Goldinger, D.M., Anne-Laure Demierre a and Otmar Zoller b, H. R., 2015. Endocrine activity of alternatives to BPA found in thermal paper in Switzerland. Regul. Toxicol. Pharmacol. 71, 453-462.
Grimaldi, M., Boulahtouf, A., Toporova, L., Balaguer, P., 2019. Functional profiling of bisphenols for nuclear receptors. Toxicology 420, 39-45.
Hecker, M., Giesy, J.P., 2008. Novel trends in endocrine disruptor testing: the H295R Steroidogenesis Assay for identification of inducers and inhibitors of hormone production. Anal. Bioanal. Chem. 390, 287-291.
Hecker, M., Newsted, J.L., Murphy, M.B., Higley, E.B., Jones, P.D., Wu, R., Giesy, J.P., 2006. Human adrenocarcinoma (H295R) cells for rapid in vitro determination of effects on steroidogenesis: hormone production. Toxicol. Appl. Pharmacol. 217, 114-124.
Jia, Z., Wang, H., Feng, Z., Zhang, S., Wang, L., Zhang, J., Liu, Q., Zhao, X., Feng, D., Feng, X., 2019. Fluorene-9-bisphenol exposure induces cytotoxicity in mouse oocytes and causes ovarian damage. Ecotoxicol. Environ. Saf. 180, 168-178.
Jiao, X., Ding, Z., Meng, F., Zhang, X., Wang, Y., Chen, F., Duan, Z., Wu, D., Zhang, S., Miao, Y., Huo, L., 2020. The toxic effects of Fluorene-9-bisphenol on porcine oocyte in vitro maturation. Environ. Toxicol. 35, 152-158.
Jiao, X.F., Liang, Q.M., Wu, D., Ding, Z.M., Zhang, J.Y., Chen, F., Wang, Y.S., Zhang, S.X., Miao, Y.L., Huo, L.J., 2019. Effects of acute fluorene-9-bisphenol exposure on mouse oocyte in vitro maturation and its possible mechanisms. Environ. Mol. Mutagen 60, 243-253.
Jin, H., Zhu, L., 2016. Occurrence and partitioning of bisphenol analogues in water and sediment from Liaohe River Basin and Taihu Lake, China. Water Res 103, 343-351.
Jin, W., Ma, R., Zhai, L., Xu, X., Lou, T., Huang, Q., Wang, J., Zhao, D., Li, X., Sun, L., 2020. Ginsenoside Rd attenuates ACTH-induced corticosterone secretion by blocking the MC2R-CAMP/PKA/CREB pathway in Y1 mouse adrenocortical cells. Life Sci. 245, 117337.
Karrer, C., de Boer, W., Delmaar, C., Cai, Y., Crepet, A., Hungerbuhler, K., von Goetz, N., 2019. Linking probabilistic exposure and pharmacokinetic modeling to assess the cumulative risk from the bisphenols BPA, BPS, BPF, and BPAF for Europeans. Environ. Sci. Technol. 53, 9181-9191.
Li, Z., Yin, N., Liu, Q., Wang, C., Wang, T., Wang, Y., Qu, G., Liu, J., Cai, Y., Zhou, Q., Jiang, G., 2013. Effects of polycyclic musks HHCB and AHTN on steroidogenesis in H295R cells. Chemosphere 90, 1227-1235.
Liu, J., Zeng, L., Zhuang, S., Zhang, C., Li, Y., Zhu, J., Zhang, W., 2020. Cadmium exposure during prenatal development causes progesterone disruptors in multiple generations via steroidogenic enzymes in rat ovarian granulosa cells. Ecotoxicol. Environ. Saf. 201, 110765.
Liu, W., Wang, J., Qiu, Q.H., Ji, L., Wang, C.Y., Zhang, M.L., 2008. Synthesis and characterisation of 9,9-bis(4-hydroxyphenyl)-fluorene catalysed by cation exchanger. Pigment Resin Technol. 37, 9-15.
Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real- time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408. Medwid, S., Guan, H., Yang, K., 2016. Prenatal exposure to bisphenol A disrupts adrenal steroidogenesis in adult mouse offspring. Environ. Toxicol. Pharmacol. 43, 203-208.
Meinsohn, M.C., Smith, O.E., Bertolin, K., Murphy, B.D., 2019. The orphan nuclear receptors steroidogenic factor-1 and liver receptor homolog-1: structure, regulation, and essential roles in mammalian reproduction. Physiol. Rev. 99, 1249-1279.
Mi, P., Zhang, Q.P., Zhang, S.H., Wang, C., Zhang, S.Z., Fang, Y.C., Gao, J.Z., Feng, D.F., Chen, D.Y., Feng, X.Z., 2019. The effects of fluorene-9-bisphenol on female zebrafish (Danio rerio) reproductive and exploratory behaviors. Chemosphere 228, 398-411. Nogueira, E.F., Rainey, W.E., 2010. Regulation of aldosterone synthase by activator transcription factor/cAMP response element-binding protein family members. Endocrinology 151, 1060-1070.
Oskarsson, A., Ulleras, E., Plant, K.E., Hinson, J.P., Goldfarb, P.S., 2006. Steroidogenic gene expression in H295R cells and the human adrenal gland: adrenotoxic effects of lindane in vitro. J. Appl. Toxicol. 26, 484-492.
Rahman, M.S., Kwon, W.S., Karmakar, P.C., Yoon, S.J., Ryu, B.Y., Pang, M.G., 2017. Gestational exposure to bisphenol a affects the function and proteome profile of F1 spermatozoa in adult mice. Environ. Health Perspect. 125, 238-245.
Rosenmai, A.K., Dybdahl, M., Pedersen, M., Vugt-Lussenburg, B.M.A. v, Wedebye, E.B., Taxvig, C., Vinggaard, A.M., 2014. Are structural analogues to bisphenol A safe alternatives? Toxicol. Sci. 139, 35-47.
Smith, L.I.F., Huang, V., Olah, M., Trinh, L., Liu, Y., Hazell, G., Conway-Campbell, B., Zhao, Z., Martinez, A., Lefrancois-Martinez, A.M., Lightman, S., Spiga, F., Aguilera, G., 2020. Involvement of CREB-regulated transcription coactivators (CRTC) in transcriptional activation of steroidogenic acute regulatory protein (Star) by ACTH. Mol. Cell. Endocrinol. 499, 110612.
Song, S., Duan, Y., Zhang, T., Zhang, B., Zhao, Z., Bai, X., Xie, L., He, Y., Ouyang, J.P., Huang, X., Sun, H., 2019. Serum concentrations of bisphenol A and its alternatives in elderly population living around e-waste recycling facilities in China: associations with fasting blood glucose. Ecotoxicol. Environ. Saf. 169, 822-828.
Wan, X., Ru, Y., Chu, C., Ni, Z., Zhou, Y., Wang, S., Zhou, Z., Zhang, Y., 2016. Bisphenol A accelerates capacitation-associated protein tyrosine phosphorylation of rat sperm by activating protein kinase A. Acta Biochim Biophys. Sin. (Shanghai) 48, 573-580.
Wang, C., Ruan, T., Liu, J., He, B., Zhou, Q., Jiang, G., 2015. Perfluorooctyl iodide stimulates steroidogenesis in H295R cells via a cyclic adenosine monophosphate signaling pathway. Chem. Res. Toxicol. 28, 848-854.
Wang, L., Zhuang, T., Li, F., Wei, W., 2019. Fluorene-9-bisphenol inhibits epithelial- mesenchymal transition of human endometrial cancer Ishikawa cells by repressing TGF-beta signaling pathway. Environ. Sci. Pollut. Res Int 26, 27407-27413.
Wu, D., Wu, F., Lin, R., Meng, Y., Wei, W., Sun, Q., Jia, L., 2020. Impairment of learning and memory induced by perinatal exposure to BPA is associated with ERalpha- mediated alterations of synaptic plasticity and PKC/ERK/CREB signaling pathway in offspring rats. Brain Res. Bull. 161, 43-54.
Xue, Q., Liu, X., Liu, X.C., Pan, W.X., Fu, J.J., Zhang, A.Q., 2019. The effect of structural diversity on ligand specificity and resulting signaling differences of estrogen receptor alpha. Chem. Res. Toxicol. 32, 1002-1013.
Yan, X., He, B., Liu, L., Qu, G., Shi, J., Liao, C., Hu, L., Jiang, G., 2018. Organotin exposure stimulates steroidogenesis in H295R Cell via cAMP pathway. Ecotoxicol. Environ. Saf. 156, 148-153.
Zhang, Hong, Chang, Steve, Wiseman, Yuhe, He, Higley, Eric, 2011. Bisphenol a disrupts steroidogenesis in human H295R Cells. Toxicol. Sci. 1-31.
Zhang, S., Sun, B., Wang, D., Liu, Y., Li, J., Qi, J., Zhang, Y., Bai, C., Liang, S., 2021. Chlorogenic acid ameliorates damage induced by fluorene-9-bisphenol in porcine sertoli cells. Front Pharm. 12, 678772.
Zhang, Z., Hu, Y., Guo, J., Yu, T., Sun, L., Xiao, X., Zhu, D., Nakanishi, T., Hiromori, Y., Li, J., Fan, X., Wan, Y., Cheng, S., Li, J., Guo, X., Hu, J., 2017. Fluorene-9-bisphenol is anti-oestrogenic and may cause adverse pregnancy outcomes in mice. Nat. Commun. 8, 14585.
Zhen, D., Li, Y., Yang, S., Ning, Z., Zhang, X., Jian, X., 2009. Kinetics and thermal properties of epoxy resins based on bisphenol fluorene structure. Eur. Polym. J. 45, 1941-1948.
Zheng, W., Jefcoate, C.R., 2005. Steroidogenic factor-1 interacts with cAMP response element-binding protein to mediate cAMP stimulation of CYP1B1 via a far upstream enhancer. Mol. Pharmacol. 67, 499-512.