Bisphenol-A analogue (bisphenol-S) exposure alters female reproductive tract and apoptosis/oxidative gene expression in blastocyst-derived cells

Document Type : Original Article


1 Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

2 Department of Theriogenology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

3 Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran


Objective(s): One of the major endocrine-disrupting chemicals, Bisphenol-S (BPS) has replaced bisphenol-A due to public health anxiety. The present study evaluated low dosage BPS effect on female reproductive potential, hormonal disruption, and gene expression pathways of blastocyst-derived cells.
Materials and Methods: NMRI female mice (5-6 weeks) in the estrous stage were chosen following vaginal smear examination for estrus cycle detection and BPS (0, 1, 5, 10, 50 and 100 µg/kg) was administrated subcutaneously for twenty-one consecutive days. After the last administration, blood, ovary tissue and oocytes were collected for further examination.
Results: BPS induced oxidative stress in ovarian tissue and reduced hormonal status, LH and FSH, even at low concentration. Furthermore, apoptosis was induced in blastocyst derived cells in BPS administrated mice groups even at low BPS concertation, however, P53 and E2f1 expression were downregulated in doses more than 50 µg/kg, which might indicate apoptosis pathway exchange from P53 dependent to p53 independent pathways. IVF outcome was negatively associated with blastocyst apoptosis gene expression, estrogen receptor beta (ERβ) as well as oxidative status in ovaries. Finally, Stepwise regression indicated that E2f1, Nrf2, catalase (CAT), and Malondialdehyde (MDA) could be chosen as predictor values for hatch percentage in IVF outcome.
Conclusion: In summary, this study revealed BPS might have detrimental potential in the female reproductive system by oxidation induction and hormonal alteration as well as next generation blastocyst derived cells apoptosis induction. Further studies are recommended for public health assurance of BPS safety especially for female consumed products.


1. Sengupta P, Banerjee R. Environmental toxins: Alarming impacts of pesticides on male fertility. Hum Exp Toxicol 2014; 33:1017-1039.
2.    Maqbool F, Mostafalou S, Bahadar H, Abdollahi M. Review of endocrine disorders associated with environmental toxicants and possible involved mechanisms. Life Sci 2016; 145:265-273.
3.    Hiroi H, Tsutsumi O, Momoeda M, Takai Y, Osuga Y, Taketani Y. Differential interactions of bisphenol A and17β-estradiol with estrogen receptor α (ERα) and ERβ. Endocrine J 1999; 46:773-778.
4.    Guignard D, Gayrard V, Lacroix M, Puel S, Picard-Hagen N, Viguié C. Evidence for bisphenol A-induced disruption of maternal thyroid homeostasis in the pregnant ewe at low level representative of human exposure. Chemosphere 2017; 182:458-467.
5.    Park C, Choi W, Hwang M, Lee Y, Kim S, Yu S, et al. Associations between urinary phthalate metabolites and bisphenol A levels, and serum thyroid hormones among the Korean adult population-Korean National Environmental Health Survey (KoNEHS) 2012–2014. Sci Total Environ 2017; 584:950-957.
6.    Leranth C, Hajszan T, Szigeti-Buck K, Bober J, MacLusky NJ. Bisphenol A prevents the synaptogenic response to estradiol in hippocampus and prefrontal cortex of ovariectomized nonhuman primates. Proc Natl Acad Sci 2008; 105:14187-14191.
7.    Liang H, Xu W, Chen J, Shi H, Zhu J, Liu X, et al. The association between exposure to environmental Bisphenol- A and gonadotropic hormone levels among men. PloS one 2017; 12: e0169217.
8.    Ehrlich S, Williams PL, Missmer SA, Flaws JA, Berry KF, Calafat AM, et al. Urinary bisphenol A concentrations and implantation failure among women undergoing in vitro fertilization. Environmental health perspectives. Environ Health Persp 2012; 120:978-983.
9.    Ramakrishnan S, Wayne NL. Impact of bisphenol-A on early embryonic development and reproductive maturation. Reprod Toxicol 2008; 25:177-183.
10.    Alonso-Magdalena P, García-Arévalo M, Quesada I, Nadal Á. Bisphenol-A treatment during pregnancy in mice: a new window of susceptibility for the development of diabetes in mothers later in life. Endocrinology 2015; 156:1659-1670.
11.    Cao Y, Qu X, Ming Z, Yao Y, Zhang Y. The correlation between exposure to BPA and the decrease of the ovarian reserve. Int J Clin Exp Pathol 2018; 11:3375-3382.
12.    Zhang R, Liu R, Zong W. Bisphenol S Interacts with Catalase and Induces Oxidative Stress in Mouse Liver and Renal Cells. J Agr Food Chem 2016; 64:6630-6640.
13.    Ullah H, Jahan S, Ain QU, Shaheen G, Ahsan N. Effect of bisphenol S exposure on male reproductive system of rats: A histological and biochemical study. Chemosphere 2016; 152:383-391.
14.    Kabuto H, Amakawa M, Shishibori T. Exposure to bisphenol A during embryonic/fetal life and infancy increases oxidative injury and causes underdevelopment of the brain and testis in mice. Life Sci 2004; 74:2931-2940.
15.    Kadasala NR, Narayanan B, Liu Y. International trade regulations on BPA: Global health and economic implications. Asian Dev Pol Rev 2016; 4:134-142.
16.    Del Moral LI, Le Corre L, Poirier H, Niot I, Truntzer T, Merlin J-F, et al. Obesogen effects after perinatal exposure of 4, 4′-sulfonyldiphenol (Bisphenol S) in C57BL/6 mice.  Toxicology 2016; 357-358:11-20.
17.    Wan Y, Xia W, Yang S, Pan X, He Z, Kannan K. Spatial distribution of bisphenol S in surface water and human serum from Yangtze River watershed, China: Implications for exposure through drinking water. Chemosphere 2018; 199:595-602.
18.    Thayer KA, Taylor KW, Garantziotis S, Schurman SH, Kissling GE, Hunt D, et al. Bisphenol A, bisphenol S, and 4-hydroxyphenyl 4-isoprooxyphenylsulfone (BPSIP) in urine and blood of cashiers. Environ Health Perspect 2016; 124:437-444.
19.    Liao C, Liu F, Alomirah H, Loi VD, Mohd MA, Moon HB, et al. Bisphenol S in urine from the United States and seven Asian countries: occurrence and human exposures. Environ Sci Technol 2012; 46:6860-6866.
20.    Rezg R, Abot A, Mornagui B, Aydi S, Knauf C. Effects of Bisphenol S on hypothalamic neuropeptides regulating feeding behavior and apelin/APJ system in mice. Ecotox Environ Safe 2018; 161:459-466.
21.    Hill CE, Sapouckey SA, Suvorov A, Vandenberg LN. Developmental exposures to bisphenol S, a BPA replacement, alter estrogen-responsiveness of the female reproductive tract: A pilot study. Cogent Med 2017; 4:1317690.
22.    Catanese MC, Vandenberg LN. Bisphenol S (BPS) alters maternal behavior and brain in mice exposed during pregnancy/lactation and their daughters. Endocrinology 2016; 158:516-530.
23.    LaPlante CD, Catanese MC, Bansal R, Vandenberg LN. Bisphenol S alters the lactating mammary gland and nursing behaviors in mice exposed during pregnancy and lactation. Endocrinology 2017; 158:3448-3461.
24.    Kinch CD, Ibhazehiebo K, Jeong J-H, Habibi HR, Kurrasch DM. Low-dose exposure to bisphenol A and replacement bisphenol S induces precocious hypothalamic neurogenesis in embryonic zebrafish. Proc Natl Acad Sci 2015; 112:1475-1480.
25.    Qiu W, Zhao Y, Yang M, Farajzadeh M, Pan C, Wayne NL. Actions of bisphenol A and bisphenol S on the reproductive neuroendocrine system during early development in zebrafish. Endocrinology 2015; 157:636-647.
26.    Rochester JR, Bolden AL. Bisphenol S and F: a systematic review and comparison of the hormonal activity of bisphenol A substitutes. Environ Health Perspect 2015; 123:643-650.
27.    Ji K, Hong S, Kho Y, Choi K. Effects of bisphenol S exposure on endocrine functions and reproduction of zebrafish. Environ Sci Technol 2013; 47:8793-8800.
28.    Wang W, Zhang X, Wang Z, Qin J, Wang W, Tian H, et al. Bisphenol S induces obesogenic effects through deregulating lipid metabolism in zebrafish (Danio rerio) larvae. Chemosphere 2018; 199:286-296.
29.    Liu J, Li J, Wu Y, Zhao Y, Luo F, Li S, et al. Bisphenol A metabolites and bisphenol S in paired maternal and cord serum. Environ Sci Technol 2017; 51:2456-2463.
30.    Fol VL, Brion F, Hillenweck A, Perdu E, Bruel S, Aït-Aïssa S, et al. Comparison of the in vivo biotransformation of two emerging estrogenic contaminants, BP2 and BPS, in zebrafish embryos and adults. Int J Mol Sci 2017; 18:704.
31.    Žalmanová T, Hošková K, Nevoral J, Adámková K, Kott T, Šulc M, et al. Bisphenol S negatively affects the meotic maturation of pig oocytes. Sci Rep 2017; 7:1-1
32.    Ullah H, Ambreen A, Ahsan N, Jahan S. Bisphenol S induces oxidative stress and DNA damage in rat spermatozoa in vitro and disrupts daily sperm production in vivo. Toxicol Environ Chem 2017; 99:953-965.
33.    Shi M, Sekulovski N, MacLean JA, Hayashi K. Prenatal exposure to bisphenol A analogues on male reproductive functions in mice. Toxicol Sci 2018; 163:620-631.
34.    Ok S, Kang JS, Kim KM. Cultivated wild ginseng extracts upregulate the anti-apoptosis systems in cells and mice induced by bisphenol A. Mol Cell Toxicol 2017; 13:73-82.
35.    Yin L, Dai Y, Cui Z, Jiang X, Liu W, Han F, et al. The regulation of cellular apoptosis by the ROS-triggered PERK/EIF2α/chop pathway plays a vital role in bisphenol A-induced male reproductive toxicity. Toxicol Appl Pharm 2017; 314:98-108.
36.    Nourian A, Soleimanzadeh A, Jalali AS, Najafi G. Effects of bisphenol-S low concentrations on oxidative stress status and in vitro fertilization potential in mature female mice. Vet Res Forum 2017: 8:341-345.
37.    Nevoral J, Kolinko Y, Moravec J, Zalmanova T, Hoskova K, Prokesova S, et al. Long-term exposure to very low doses of bisphenol S affects female reproduction. Reproduction 2018: 156:47-57.
38.    Byers SL, Wiles MV, Dunn SL, Taft RA. Mouse estrous cycle identification tool and images. PLoS one 2012; 7:e35538.
39.    Rubin BS. Bisphenol A: an endocrine disruptor with widespread exposure and multiple effects. J Steroid Biochem Mol Biol 2011; 127:27-34.
40.    Babaei M, Najafi G, Jalali AS, Behfar M. Effects of unilateral iatrogenic vas deferens trauma on fertility: An experimental in vitro fertilization mice model study. Bull Emerg Trauma 2015; 3:122-127.
41.    Choi BI, Harvey AJ, Green MP. Bisphenol A affects early bovine embryo development and metabolism that is negated by an oestrogen receptor inhibitor. Sci Rep 2016; 6:29318-29337.
42.    Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem 1996; 239:70-76.
43.    Nishikimi M, Rao NA, Yagi K. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Bioph Res Co 1972; 46:849-854.
44.    Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70:158-169.
45.    Sinha AK. Colorimetric assay of catalase. Anal Biochem 1972; 47:389-394.
46.    Pell T, Eliot M, Chen A, Lanphear BP, Yolton K, Sathyanarayana S, et al. Parental concern about environmental chemical exposures and children’s urinary concentrations of phthalates and phenols. J Pediatr 2017; 186:138-144.
47.    Zhou X, Kramer JP, Calafat AM, Ye X. Automated on-line column-switching high performance liquid chromatography isotope dilution tandem mass spectrometry method for the quantification of bisphenol A, bisphenol F, bisphenol S, and 11 other phenols in urine. J Chromatogr B 2014; 944:152-156.
48.    Björnsdotter MK, de Boer J, Ballesteros-Gómez A. Bisphenol A and replacements in thermal paper: A review. Chemosphere 2017; 182:691-706.
49.    Fernández M, Bourguignon N, Lux-Lantos V, Libertun C. Neonatal exposure to bisphenol a and reproductive and endocrine alterations resembling the polycystic ovarian syndrome in adult rats. Environ Health Perspect 2010; 118:1217-1222.
50.    Jin P, Wang X, Chang F, Bai Y, Li Y, Zhou R, et al. Low dose bisphenol A impairs spermatogenesis by suppressing reproductive hormone production and promoting germ cell apoptosis in adult rats. J Biomed Res 2013; 27:135-144.
51.    Vahedi M, Saeedi A, Poorbaghi SL, Sepehrimanesh M, Fattahi M. Metabolic and endocrine effects of bisphenol A exposure in market seller women with polycystic ovary syndrome. Environ Sci Pollut Res 2016; 23:23546-23550.
52.    Ma S, Shi W, Wang X, Song P, Zhong X. Bisphenol-A exposure during pregnancy alters the Mortality and Levels of Reproductive Hormones and Genes in Offspring Mice. BioMed Res Int 2017; 2017.
53.    Patel S, Brehm E, Gao L, Rattan S, Ziv-Gal A, Flaws JA. Bisphenol-A exposure, ovarian follicle numbers, and female sex steroid hormone levels: Results from a CLARITY-BPA study. Endocrinology 2017; 158:1727-1738.
54.    Ogo FM, Siervo GE, Gonçalves GD, Cecchini R, Guarnier FA, Anselmo-Franci JA, et al. Low doses of bisphenol A can impair postnatal testicular development directly, without affecting hormonal or oxidative stress levels. Reprod Fert Develop 2017; 29:2245-2254.
55.    Ahsan N, Ullah H, Ullah W, Jahan S. Comparative effects of Bisphenol S and Bisphenol A on the development of female reproductive system in rats; a neonatal exposure study. Chemosphere 2018; 197:336-343.
56.    Yazdani M, Andresen AMS, Gjøen T. Short-term effect of bisphenol-a on oxidative stress responses in Atlantic salmon kidney cell line: a transcriptional study. Toxicol Mech Method 2016; 26:295-300.
57.    Huc L, Lemarié A, Guéraud F, Héliès-Toussaint C. Low concentrations of bisphenol A induce lipid accumulation mediated by the production of reactive oxygen species in the mitochondria of HepG2 cells. Toxicol in vitro 2012; 26:709-717.
58.    Maćczak A, Cyrkler M, Bukowska B, Michałowicz J. Bisphenol A, bisphenol S, bisphenol F and bisphenol AF induce different oxidative stress and damage in human red blood cells (in vitro study). Toxicol in vitro 2017; 41:143-149.
59.    Desdoits-Lethimonier C, Lesné L, Gaudriault P, Zalko D, Antignac J, Deceuninck Y, et al.  Parallel assessment of the effects of bisphenol A and several of its analogs on the adult human testis. Hum Reprod 2017: 32:1465-1473.
60.    Mobley JA, Brueggemeier RW. Estrogen receptor-mediated regulation of oxidative stress and DNA damage in breast cancer. Carcinogenesis 2004; 25:3-9.
61.    Nadal A, Fuentes E, Ripoll C, Villar-Pazos S, Castellano-Muñoz M, Soriano S, et al. Extranuclear-initiated estrogenic actions of endocrine disrupting chemicals: Is there toxicology beyond paracelsus? J Steroid Biochem Mol Biol 2018; 176:16-22.
62.    Bredhult C, Bäcklin B-M, Olovsson M. Effects of some endocrine disruptors on the proliferation and viability of human endometrial endothelial cells in vitro. Reprod toxicol 2007; 23:550-559.
63.    Ptak A, Wróbel A, Gregoraszczuk EL. Effect of bisphenol-A on the expression of selected genes involved in cell cycle and apoptosis in the OVCAR-3 cell line. Toxicol lett 2011; 202:30-35.
64.    Sengupta S, Obiorah I, Maximov P, Curpan R, Jordan V. Molecular mechanism of action of bisphenol and bisphenol A mediated by oestrogen receptor alpha in growth and apoptosis of breast cancer cells.  Br J Pharmacol 2013; 169:167-178.
65.    Rosenmai AK, Dybdahl M, Pedersen M, Alice van Vugt-Lussenburg BM, Wedebye EB, Taxvig C, et al. Are structural analogues to bisphenol a safe alternatives? Toxicol Sci 2014; 139:35-47.
66.    Fic A, Žegura B, Sollner Dolenc M, Filipič M, Peterlin Mašič L. Mutagenicity and DNA damage of bisphenol A and its structural analogues in HepG2 cells. Arh Hig Rada Toksikol 2013; 64:189-200.
67.    Choi J, Donehower L. p53 in embryonic development: maintaining a fine balance. Cell Mol Life Sci 1999; 55:38-47.
68.    Bondesson M, Hao R, Lin C-Y, Williams C, Gustafsson J-Å. Estrogen receptor signaling during vertebrate development. Biochim Biophys Acta 2015; 1849:142-151.
69.    Lemmen JG, Broekhof JL, Kuiper GG, Gustafsson J-Å, Van Der Saag PT, Van Der Burg B. Expression of estrogen receptor alpha and beta during mouse embryogenesis. Mech Dev 1999; 81:163-167.
70.    Ramos JG, Varayoud J, Kass L, Rodríguez H, Costabel L, Muñoz-de-Toro Mn, et al. Bisphenol A induces both transient and permanent histofunctional alterations of the hypothalamic-pituitary-gonadal axis in prenatally exposed male rats. Endocrinology 2003; 144:3206-3215.
71.    Susiarjo M, Hassold TJ, Freeman E, Hunt PA. Bisphenol A exposure in utero disrupts early oogenesis in the mouse. PLoS genet 2007; 3:e5.
72.    Hou Q, Gorski J. Estrogen receptor and progesterone receptor genes are expressed differentially in mouse embryos during preimplantation development. Proc Natl Acad Sci 1993; 90:9460-9464.
73.    Saito K, Furukawa E, Kobayashi M, Fukui E, Yoshizawa M, Matsumoto H. Degradation of estrogen receptor α in activated blastocysts is associated with implantation in the delayed implantation mouse model. Mol Hum Reprod 2014; 20:384-391.
74.    Qiu W, Yang M, Liu S, Lei P, Hu L, Chen B, et al. Toxic effects of bisphenol S showing immunomodulation in fish macrophages. Environ Sci Technol 2018; 52:831-838.
75.    Viñas R, Watson CS. Mixtures of xenoestrogens disrupt estradiol-induced non-genomic signaling and downstream functions in pituitary cells. Environ Health 2013; 12:26-36.
76.    Li Y, Perera L, Coons LA, Burns KA, Ramsey JT, Pelch KE, et al. Differential in vitro biological action, coregulator interactions, and molecular dynamic analysis of Bisphenol-A (BPA), BPAF, and BPS ligand–ERα complexes. Environ Health 2018; 126:017012.
77.    Palena A, Mangiacasale R, Magnano AR, Barberi L, Giordano R, Spadafora C, et al. E2F transcription factors are differentially expressed in murine gametes and early embryos. Mech Dev 2000; 97:211-215.
78.    Liao Y, Du W. Rb‐independent E2F3 promotes cell proliferation and alters expression of genes involved in metabolism and inflammation. FEBS Open Bio 2017; 7:1611-1621.
79.    Yamasaki L, Bronson R, Williams BO, Dyson NJ, Harlow E, Jacks T. Loss of E2F-1 reduces tumorigenesis and extends the lifespan of Rb1 (+/−) mice. Nat Genet 1998; 18:360-364.
80.    Ziebold U, Lee EY, Bronson RT, Lees JA. E2F3 loss has opposing effects on different pRB-deficient tumors, resulting in suppression of pituitary tumors but metastasis of medullary thyroid carcinomas. Mol Cell Biol 2003; 23:6542-6552.
81.    Cheraghi S, Razi M, Malekinejad H. Involvement of cyclin D1 and E2f1 in zearalenone-induced DNA damage in testis of rats. Toxicon 2015; 106:108-116.
82.    Zhan L, Zhang Y, Wang W, Song E, Fan Y, Wei B. E2F1: a promising regulator in ovarian carcinoma. Tumor Biol 2016; 37:2823-2831.
83.    Cayirlioglu P, Ward WO, Key SCS, Duronio RJ. Transcriptional repressor functions of Drosophila E2F1 and E2F2 cooperate to inhibit genomic DNA synthesis in ovarian follicle cells. Mol Cell Biol 2003; 23:2123-2134.
84.    Roos W, Christmann M, Fraser S, Kaina B. Mouse embryonic stem cells are hypersensitive to apoptosis triggered by the DNA damage O6-methylguanine due to high E2F1 regulated mismatch repair. Cell Death Differ 2007; 14:1422-1432.
85.    Pfeifer D, Chung YM, Hu MC. Effects of low-dose bisphenol A on DNA damage and proliferation of breast cells: the role of c-Myc. Environ Health Perspect 2015; 123:1271-1279.
86.    Liao XH, Lu DL, Wang N, Liu LY, Wang Y, Li YQ, et al. Estrogen receptor α mediates proliferation of breast cancer MCF–7 cells via a p21/PCNA/E2F1‐dependent pathway. FEBS J 2014; 281:927-942.
87.    Wang W, Dong L, Saville B, Safe S. Transcriptional activation of E2F1 gene expression by 17β-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions. Mol Endocrinol 1999; 13:1373-1387.
88.    Frietze S, Lupien M, Silver PA, Brown M. CARM1 regulates estrogen-stimulated breast cancer growth through up-regulation of E2F1. Cancer Res 2008; 68:301-306.
89.    Louie MC, McClellan A, Siewit C, Kawabata L. Estrogen receptor regulates E2F1 expression to mediate tamoxifen resistance. Mol Cancer Res 2010; 8:343-352.
90.    Lin Y, Sui LC, Wu RH, Fu HY, Xu JJ, Qiu XH, et al. Nrf2 inhibition affects cell cycle progression during early mouse embryo development. J Reprod Develop 2018;64:49-55.
91.    Kobayashi M, Yamamoto M. Molecular mechanisms activating the Nrf2-Keap1 pathway of antioxidant gene regulation. Antioxid Redox Sign 2005; 7:385-394.
92.    Gad A, Hoelker M, Besenfelder U, Havlicek V, Cinar U, Rings F, et al. Molecular mechanisms and pathways involved in bovine embryonic genome activation and their regulation by alternative in vivo and in vitro culture conditions. Biol Reprod 2012; 87:100, 1-13.
93.    Held E, Salilew-Wondim D, Linke M, Zechner U, Rings F, Tesfaye D, et al. Transcriptome fingerprint of bovine 2-cell stage blastomeres is directly correlated with the individual developmental competence of the corresponding sister blastomere. Biol Reprod 2012; 87:154, 1-13.
94.    Bomfim MM, Andrade GM, del Collado M, Sangalli JR, Fontes PK, Nogueira MF, et al. Antioxidant responses and deregulation of epigenetic writers and erasers link oxidative stress and DNA methylation in bovine blastocysts. Mol Reprod Dev 2017; 84:1296-1305.
95.    Amin A, Gad A, Salilew‐Wondim D, Prastowo S, Held E, Hoelker M, et al. Bovine embryo survival under oxidative‐stress conditions is associated with activity of the NRF2‐mediated oxidative‐stress‐response pathway. Mol Reprod Dev 2014; 81:497-513.
96.    Shimpi PC, More VR, Paranjpe M, Donepudi AC, Goodrich JM, Dolinoy DC, et al. Hepatic lipid accumulation and Nrf2 expression following perinatal and peripubertal exposure to Bisphenol-A in a mouse model of nonalcoholic liver disease. Environ Health Perspect 2017; 125:087005.