Alteration on global and gene-spesific DNA methylation and global histone modifications in HepG2 cells in response to BPA

Year 2016, Volume: 46 Issue: 2, 97 - 114, 26.12.2016

Mine Şenyıldız Ecem Fatma Karaman Serap Sancar Baş Pelin Arda Pirinççi , Sibel Özden

Abstract

Bisphenol A (BPA), as synthetic monomer used in industry in the production of polycarbonate plastic and epoxy resins, has endocrine disruptor properties and high risk on human health. Continuous release of free BPA into food, beverages, and the environment has resulted in a widespread human exposure to this chemical. Recent studies have showed the role of endocrine effects of environmental chemicals on the changes in gene expression may be associated with epigenetic mechanisms such as DNA methylation and histone modifications. The aim of the study was to investigate dose-related effects of BPA (0, 0.1 μM, 1 and 10 mM for 48 and 96 h) on global and gene-spesific (p16, cyclin D2 and Rassf1 genes) DNA methylation in human hepatocarcinoma (HepG2) cells. We also investigated global histone modifications such as H3K9 trimethylation (H3K9me3), H3K9 acetylation (H3K9ac), H3K27 trimethylation (H3K27me3). The 50% inhibitory concentration (IC50) value of BPA was determined as 134 and 180 μM in HepG2 cells for 24 h by MTT and neutral red uptake (NRU) tests, respectively. We observed decrease on the global levels of 5-methylcytosine and 5-hydroxymethylcytosine at 1 and 10 mM after 96 h BPA exposure. There is no significant alterations on the promoter-region of methylation and expression of p16, cyclin D2 and Rassf1 genes. Global levels of H3K9me3 decreased after 0.1 and 1 μM concentration of BPA exposure for 48 h, while increased after 96 h 0.1 and 1 μM of BPA exposure for. Hovewer, it has not been observed significantly changes for the global levels of H3K27me3 and H3K9ac. In this study we suggest that BPA may disrupt epigenetic events by altering global and gene-spesific DNA methylation and histone modifications in HepG2 cells.

Keywords

Bisphenol A, DNA methylation, cytotoxicity, histone modifications, HepG2 cells

References

• Agarwal S, Yadav A, Tiwari SK, Seth B, Chauhan LKS, Khare P, Ray RS, Chaturvedi RK (2016) Dynamin-related protein 1 inhibition mitigates Bisphenol-A mediated alterations in mitochondrial dynamics and neural stem cells proliferation and differentiation, J Biol Chem., 291(31): 15923-39.
• Agathanggelou A, Cooper WN, Latif F (2005) Role of the Ras-association domain family 1 tumor suppressor gene in human cancers, Cancer Res., 65(9): 3497-3508.
• Alley MC, Scudiere DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL, Abbott BJ, Mayo JG, Shoemaker RH, Boyd MR (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay, Cancer Res., 48: 589-601.
• Alonso-Magdalena P, García-Arévalo M, Quesada I, Nadal Á (2015) Bisphenol-A treatment during pregnancy in mice: a new window of susceptibility for the development of diabetes in mothers later in life, Endocrinology, 156(5): 1659-1670.
• Araújo OC, Rosa AS, Fernandes A, Niel C, Villela-Nogueira CA, Pannain V, Araujo NM (2016) RASSF1A and DOK1 promoter methylation levels in hepatocellular carcinoma, cirrhotic and non-cirrhotic liver, and correlation with liver cancer in Brazilian patients, PloS One, 11(4): e0153796.
• Belinsky SA, Nikula KJ, Palmisano WA, Michels R, Saccomanno G, Gabrielson E, Baylin SB, Herman JG (1998) Aberrant methylation of p16INK4a is an early event in lung cancer and a potential biomarker for early diagnosis, Proc Natl Acad Sci U S A., 95(20): 11891-11896.
• Bhan A, Hussain I, Ansari KI, Bobzean SA, Perrotti LI, Mandal SS (2014) Histone methyltransferase EZH2 is transcriptionally induced by estradiol as well as estrogenic endocrine disruptors bisphenol-A and diethylstilbestrol, J Mol Biol., 426(20): 3426-3441.
• Borenfreund E and Puerner JA (1985), Toxicity determined in vitro by morphological alterations and neutral red absorption, Toxicol Lett., 24(2): 119-124.
• Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem., 72(1-2): 248-254.
• Burbee DG, Forgacs E, Zöchbauer-Müller S, Shivakumar L, Fong K, Gao B, Randle D, Kondo M, Virmani A, Bader S, Sekido Y, Latif F, Milchgrub S, Toyooka S, Gazdar AF, Lerman MI, Zabarovsky E, White M, Minna JD (2001) Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression, J Natl Cancer Inst., 93(9): 691-699.
• Choudhary C, Kumar C, Gnad F, Nielsen ML, Rehman M, Walther TC, Olsen JV, Mann M (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions, Science, 325(5942): 834-840.
• Doherty LF, Bromer JG, Zhou Y, Aldad TS, Taylor HS (2010) In utero exposure to diethylstilbestrol (DES) or bisphenol-A (BPA) increases EZH2 expression in the mammary gland: an epigenetic mechanism linking endocrine disruptors to breast cancer, Horm Cancer., 1(3): 146-155.
• EFSA (2015) Scientific Opinion on the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs: PART II - Toxicological assessment and risk characterisation, EFSA Journal, 13(1): 3978.
• Faulk C, Kim JH, Jones TR, McEachin RC, Nahar MS, Dolinoy DC, Sartor MA (2015) Bisphenol A-associated alterations in genome-wide DNA methylation and gene expression patterns reveal sequence-dependent and non-monotonic effects in human fetal liver, Environmental Epigenetics, doi: 10.1093/eep/dvv006.
• Faulk C, Kim JH, Anderson OS, Nahar MS, Jones TR, Sartor MA, Dolinoy DC (2016) Detection of Differential DNA Methylation in Repetitive DNA of Mice and Humans Perinatally Exposed to Bisphenol A, Epigenetics, 11(7): 489-500.
• 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.
• Gassman NR, Coskun E, Jaruga P, Dizdaroglu M, Wilson SH (2016) Combined Effects of High-Dose Bisphenol A and Oxidizing Agent (KBrO3) on Cellular Microenvironment, Gene Expression, and Chromatin Structure of Ku70-deficient Mouse Embryonic Fibroblasts, Environ Health Perspect., 124(8): 1241-52.
• George O, Bryant BK, Chinnasamy R, Corona C, Arterburn JB, Shuster CB (2008) Bisphenol A directly targets tubulin to disrupt spindle organization in embryonic and somatic cells, ACS Chem Biol., 3(3): 167-179.
• Geraldes C, Gonçalves AC, Cortesão E, Pereira MI, Roque A, Paiva A, Sarmento-Ribeiro AB (2016) Aberrant p15, p16, p53, and DAPK Gene Methylation in Myelomagenesis: Clinical and Prognostic Implications. Clinical Lymphoma Myeloma and Leukemia,. doi: 10.1016/j.clml.2016.08.016.
• Greathouse KL, Bredfeldt T, Everitt JI, Lin K, Berry T, Kannan K, Mittelstadt ML, Ho SM, Walker CL (2012) Environmental estrogens differentially engage the histone methyltransferase EZH2 to increase risk of uterine tumorigenesis, Mol Cancer Res., 10(4): 546-557.
• Ho SM, Cheong A, Lam HM, Hu WY, Shi GB, Zhu X, Chen J, Zhang X, Medvedovic M, Leung YK, Prins GS (2015) Exposure of Human Prostaspheres to Bisphenol A Epigenetically Regulates SNORD Family Noncoding RNAs via Histone Modification, Endocrinology, 156(11): 3984-3995.
• Huang Y, Pastor WA, Shen Y, Tahiliani M, Liu DR, Rao A (2010) The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing, PloS One, 5(1): e8888.
• Jueliger S, Lyons J, Cannito S, Pata I, Pata P, Shkolnaya M, Lo Re O, Peyrou M, Villarroya F, Pazienza V, Rappa F, Cappello F, Azab M, Taverna P, Vinciguerra M (2016) Efficacy and epigenetic interactions of novel DNA hypomethylating agent guadecitabine (SGI-110) in preclinical models of hepatocellular carcinoma, Epigenetics, 11: 1-12.
• Ke ZH, Pan JX, Jin LY, Xu HY, Yu TT, Ullah K, Rahman TU, Ren J, Cheng Y, Dong XY, Sheng JZ, Huang HF (2016) Bisphenol A Exposure May Induce Hepatic Lipid Accumulation via Reprogramming the DNA Methylation Patterns of Genes Involved in Lipid Metabolism, Scientific Reports, doi:10.1038/srep31331.
• Klacz J, Wierzbicki PM, Wronska A, Rybarczyk A, Stanislawowski M, Slebioda T, Kmiec Z (2016) Decreased expression of RASSF1A tumor suppressor gene is associated with worse prognosis in clear cell renal cell carcinoma, Int J Oncol., 48(1): 55-66.
• Kufe DW, Pollock RE, Weichselbaum RR, Bast RC, Gansler TS, Holland JF, Frei E (2003). Cancer medicine 6. 6th ed, BC Decker, Inc., USA.
• Laing LV, Viana J, Dempster EL, Trznadel M, Trunkfield LA, Webster TU, van Aerle R, Paull GC, Wilson RJ, Mill J, Santos EM (2016) Bisphenol A causes reproductive toxicity, decreases dnmt1 transcription, and reduces global DNA methylation in breeding zebrafish (Danio rerio), Epigenetics, 11(7): 526-38.
• Liao WT, Lu JH, Lee CH, Lan CCE, Chang JG, Chai CY, Yu HS (2016) An Interaction between Arsenic-Induced Epigenetic Modification and Inflammatory Promotion in a Skin Equivalent during Arsenic Carcinogenesis, J Invest Dermatol, doi: 10.1016/j. jid.2016.08.017.
• Liggett WH, Sidransky D (1998) Role of the p16 tumor suppressor gene in cancer, J Clin Oncol., 16(3): 1197-1206.
• Ling Q, Shi W, Huang C, Zheng J, Cheng Q, Yu K, Chen S, Zhang H, Li N, Chen M (2014) Epigenetic silencing of dual oxidase 1 by promoter hypermethylation in human hepatocellular carcinoma, Am J Cancer Res., 4: 508-517.
• Lübbert M and Jones PA (2014) Epigenetic Therapy of Cancer. 1st ed, Springer-Verlag Berlin Heidelberg, Germany.
• Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J Immunol Methods., 65(1–2): 55-63.
• Nakagawa Y, Tayama S (2000) Metabolism and cytotoxicity of bisphenol A and other bisphenols in isolated rat hepatocytes, Arch Toxicol., 74(2): 99-105.
• Pertega-Gomes N, Vizcaino JR, Felisbino S, Warren AY, Shaw G, Kay J, Whitaker H, Lynch AG, Fryer L, Neal DE, Massie CE (2015) Epigenetic and oncogenic regulation of SLC16A7 (MCT2) results in protein over-expression, impacting on signalling and cellular phenotypes in prostate cancer, Oncotarget, 6(25): 21675-84.
• Peters I, Rehmet K, Wilke N, Kuczyk MA, Hennenlotter J, Eilers T, Serth J (2007) RASSF1A promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis. Mol Cancer., 6: 49.
• Pu RT, Laitala LE, Clark DP (2006) Methylation profiling of urothelial carcinoma in bladder biopsy and urine, Acta Cytol., 50(5): 499-506.
• Qin XY, Zaha H, Nagano R, Yoshinaga J, Yonemoto J, Sone H (2011) Xenoestrogens down-regulate aryl-hydrocarbon receptor nuclear translocator 2 mRNA expression in human breast cancer cells via an estrogen receptor alpha-dependent mechanism, Toxicol Lett., 206(2): 152-7.
• Qin XY, Fukuda T, Yang L, Zaha H, Akanuma H, Zeng Q, Yoshinaga J, Sone H (2012) Effects of bisphenol A exposure on the proliferation and senescence of normal human mammary epithelial cells, Cancer Biol Ther., 13(5): 296-306.
• Repetto G, del Peso A, Zurita JL (2008) Neutral red uptake assay for the estimation of cell viability/cytotoxicity, Nat Protoc., 3(7): 1125-1131.
• Rezg R, El-Fazaa S, Gharbi N, Mornagui B (2014) Bisphenol A and human chronic diseases: current evidences, possible mechanisms, and future perspectives, Environ Int., 64: 83-90.
• Rubin BS (2011) Bisphenol A: an endocrine disruptor with widespread exposure and multiple effects, J Steroid Biochem Mol Biol., 127(1-2): 27-34.
• Saksouk N, Simboeck E, Déjardin J (2015) Constitutive heterochromatin formation and transcription in mammals, Epigenetics Chromatin, 8: 3.
• Sales LB, Kamstra JH, Cenijn PH, Van Rijt LS, Hamers T, Legler J (2013) Effects of endocrine disrupting chemicals on in vitro global DNA methylation and adipocyte differentiation, Toxicol In Vitro., 27(6): 1634-1643.
• Singh S, Li SSL (2012) Epigenetic effects of environmental chemicals bisphenol a and phthalates, Int J Mol Sci., 13(8): 10143-10153.
• Spangle JM, Dreijerink KM, Groner AC, Cheng H, Ohlson CE, Reyes J, Lin CY, Bradner J, Zhao JJ, Roberts TM, Brown M (2016) PI3K/AKT Signaling Regulates H3K4 Methylation in Breast Cancer, Cell Rep., 15(12): 2692-704.
• Şenyıldız M, Özden S (2015) Alteration in global DNA methylation after bisphenol a exposure in MCF-7 cells, J Fac Pharm Istanbul, 45(2): 153-164.
• Tabish AM, Poels K, Hoet P, Godderis L (2012) Epigenetic factors in cancer risk: effect of chemical carcinogens on global DNA methylation pattern in human TK6 cells, PLoS One, 7(4): e34674.
• Takahashi O and Oishi S (2001) Testicular toxicity of dietary 2, 2-bis (4-hydroxyphenyl) propane (bisphenol A) in F344 rats, Arch Toxicol., 75(1): 42-51.
• Toyooka S, Toyooka KO, Maruyama R, Virmani AK, Girard L, Miyajima K, Harada K, Ariyoshi Y, Takahashi T, Sugio K, Brambilla E, Gilcrease M, Minna JD, Gazdar AF (2001) DNA Methylation Profiles of Lung Tumors, Mol Cancer Ther., 1(1): 61-67.
• Udali S, Guarini P, Ruzzenente A, Ferrarini A, Guglielmi A, Lotto V, Tononi P, Pattini P, Moruzzi S, Campagnaro T, Conci S, Olivieri O, Corrocher R, Delledonne M, Choi SW, Friso S (2015) DNA methylation and gene expression profiles show novel regulatory pathways in hepatocellular carcinoma, Clin Epigenetics., 7(1): 43.
• Wang L, Cui Y, Zhang L, Sheng J, Yang Y, Kuang G, Fan Y, Zhang Q, Jin J (2016b) The Silencing of CCND2 by Promoter Aberrant Methylation in Renal Cell Cancer and Analysis of the Correlation between CCND2 Methylation Status and Clinical Features, PloS One, 11(9): e0161859.
• Wang T, Han J, Duan X, Xiong B, Cui XS, Kim NH, Liu HL, Sun SC (2016a) The toxic effects and possible mechanisms of Bisphenol A on oocyte maturation of porcine in vitro, Oncotarget, 7(22): 32554-65.
• Weinhouse C, Bergin IL, Harris C, Dolinoy DC (2015) Stat3 is a candidate epigenetic biomarker of perinatal Bisphenol A exposure associated with murine hepatic tumors with implications for human health, Epigenetics, 10(12): 1099-1110.
• Weinhouse C, Sartor MA, Faulk C, Anderson OS, Sant KE, Harris C, Dolinoy DC (2016) Epigenome wide DNA methylation analysis implicates neuronal and inflammatory signaling pathways in adult murine hepatic tumorigenesis following perinatal exposure to bisphenol A, Environ Mol Mutagen, 57(6): 435-446.
• Zeller P, Padeken J, van Schendel R, Kalck V, Tijsterman M, Gasser SM (2016) Histone H3K9 methylation is dispensable for Caenorhabditis elegans development but suppresses RNA:DNA hybrid-associated repeat instability, Nature Genetics, doi:10.1038/ng.3672.
• Zöchbauer-Müller S, Fong KM, Virmani AK, Geradts J, Gazdar AF, Minna JD (2001) Aberrant promoter methylation of multiple genes in non-small cell lung cancers, Cancer Res., 61(1): 249-255.