, Volume 70, Issue 1, pp 119–128 | Cite as

Analysis of toxicity effects of Di-(2-ethylhexyl) phthalate exposure on human bronchial epithelial 16HBE cells

  • Yue Ma
  • Yinsheng Guo
  • Shuang Wu
  • Ziquan Lv
  • Qian Zhang
  • Xing Xie
  • Yuebin KeEmail author


Recent studies have indicated that Di-(2-ethylhexyl) phthalate (DEHP), the most commonly used plasticizer in daily-life products, could be dispersed in indoor air and induce human exposure via inhalation. DEHP has been reported to have effects on the respiratory system in both animal and human researches. The toxicity effects of DEHP exposure on cell proliferation, cell cycle progression, apoptosis, global DNA methylation and the expression levels of DNA methyltransferases (DNMTs) were investigated in this study, using human epithelial cell line 16HBE as an in vitro model. Cells were treated with DEHP at doses of 0, 0.125, 0.5 and 2 mmol/L for 48 h. Cell proliferation, cell cycle and apoptosis were tested by MTT assay and flow cytometer, respectively. The obtained results showed decreased living cell number and cell viability following DEHP exposure at the dose of 2 mmol/L. DEHP also inhibited the cell cycle progression of G1 phase and induced a significant increase in cell apoptosis in 16HBE cells. DEHP exposure could induce cell proliferation inhibition in 16HBE cells via the blocking of cell cycle progression and accelerated cell apoptosis. In addition, decreased global DNA methylation levels and expression levels of DNMTs were observed in DEHP-treated groups which revealed possible epigenetic effects of DEHP.


Apoptosis Cell cycle DEHP DNA methylation Human bronchial epithelial 16HBE cell 



Di-(2-ethylhexyl) phthalate


Dimethyl sulfoxide


DNA methyltransferase 1


Fetal bovine serum


Mono-2-ethylhexyl phthalate


Minimum essential medium


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


Optical density


Phosphate buffer saline


Prodidium iodide


Polyvinyl chloride


Quantitative real-time PCR



This work was supported by funds from the National Natural Science Foundation of China (81072323, 81602831), China Postdoctoral Science Foundation funded project (2016M602537) and the Guangdong Provincial Science and Technology Project (2013B021800032). We offer our sincere thanks to all of the participants.

Compliance with ethical standards

Conflict of interest

The authors have no conflict of interest.


  1. Bestor TH (2000) The DNA methyltransferases of mammals. Hum Mol Genet 9:2395–2402CrossRefGoogle Scholar
  2. Bornehag CG, Sundell J, Weschler CJ, Sigsgaard T, Lundgren B, Hasselgren M, Hagerhed-Engman L (2004) The association between asthma and allergic symptoms in children and phthalates in house dust: a nested case-control study. Environ Health Perspect 112:1393–1397CrossRefGoogle Scholar
  3. Cadogan DF, Howick CJ (1996) Encyclopedia of chemical technology, vol. In: Othmer K (ed) Plasticizers. Wiley, New YorkGoogle Scholar
  4. Chen T, Ueda Y, Dodge JE, Wang Z, Li E (2003) Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol Cell Biol 23:5594–5605CrossRefGoogle Scholar
  5. El Kebir D, Gjorstrup P, Filep JG (2012) Resolvin E1 promotes phagocytosis-induced neutrophil apoptosis and accelerates resolution of pulmonary inflammation. Proc Natl Acad Sci USA 109:14983–14988. doi: 10.1073/pnas.1206641109 CrossRefGoogle Scholar
  6. Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516. doi: 10.1080/01926230701320337 CrossRefGoogle Scholar
  7. Glue C, Millner A, Bodtger U, Jinquan T, Poulsen LK (2002) In vitro effects of monophthalates on cytokine expression in the monocytic cell line THP-1 and in peripheral blood mononuclear cells from allergic and non-allergic donors. Toxicol in vitro: an international journal published in association with BIBRA 16:657–662CrossRefGoogle Scholar
  8. Green R, Hauser R, Calafat AM, Weuve J, Schettler T, Ringer S, Huttner K, Hu H (2005) Use of di(2-ethylhexyl) phthalate-containing medical products and urinary levels of mono(2-ethylhexyl) phthalate in neonatal intensive care unit infants. Environ Health Perspect 113:1222–1225CrossRefGoogle Scholar
  9. Guo Y, Ji J, Wang W, Dong Y, Zhang Z, Zhou Y, Chen G, Cheng J (2015) Role of endoplasmic reticulum apoptotic pathway in testicular Sertoli cells injury induced by carbon disulfide. Chemosphere 132:70–78. doi: 10.1016/j.chemosphere.2015.02.058 CrossRefGoogle Scholar
  10. Hoppin JA, Ulmer R, London SJ (2004) Phthalate exposure and pulmonary function. Environ Health Perspect 112:571–574CrossRefGoogle Scholar
  11. Hoppin JA, Jaramillo R, London SJ, Bertelsen RJ, Salo PM, Sandler DP, Zeldin DC (2013) Phthalate exposure and allergy in the U.S. population: results from NHANES 2005–2006. Environ Health Perspect 121:1129–1134. doi: 10.1289/ehp.1206211 Google Scholar
  12. Jaakkola JJ, Verkasalo PK, Jaakkola N (2000) Plastic wall materials in the home and respiratory health in young children. Am J Public Health 90:797–799CrossRefGoogle Scholar
  13. Jepsen KF, Abildtrup A, Larsen ST (2004) Monophthalates promote IL-6 and IL-8 production in the human epithelial cell line A549. Toxicology in vitro: an international journal published in association with BIBRA 18:265–269. doi: 10.1016/j.tiv.2003.09.008 CrossRefGoogle Scholar
  14. Jiang G, Xu L, Song S, Zhu C, Wu Q, Zhang L, Wu L (2008) Effects of long-term low-dose cadmium exposure on genomic DNA methylation in human embryo lung fibroblast cells. Toxicology 244:49–55. doi: 10.1016/j.tox.2007.10.028 CrossRefGoogle Scholar
  15. Kolarik B, Naydenov K, Larsson M, Bornehag CG, Sundell J (2008) The association between phthalates in dust and allergic diseases among Bulgarian children. Environ Health Perspect 116:98–103. doi: 10.1289/ehp.10498 CrossRefGoogle Scholar
  16. Laing LV, Viana J, Dempster EL, Trznadel M, Trunkfield LA, Uren Webster TM, 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 11:526–538. doi: 10.1080/15592294.2016.1182272 CrossRefGoogle Scholar
  17. Nicodemus-Johnson J, Naughton KA, Sudi J, Hogarth K, Naurekas ET, Nicolae DL, Sperling AI, Solway J, White SR, Ober C (2016) Genome-wide methylation study identifies an IL-13-induced epigenetic signature in asthmatic airways. Am J Respir Crit Care Med 193:376–385. doi: 10.1164/rccm.201506-1243OC CrossRefGoogle Scholar
  18. Pierce JD, Pierce J, Stremming S, Fakhari M, Clancy RL (2007) The role of apoptosis in respiratory diseases. Clin Nurse Spec CNS 21:22–28 quiz 29–30 CrossRefGoogle Scholar
  19. Pietenpol JA, Stewart ZA (2002) Cell cycle checkpoint signaling: cell cycle arrest versus apoptosis. Toxicology 181–182:475–481CrossRefGoogle Scholar
  20. Portela A, Esteller M (2010) Epigenetic modifications and human disease Nature biotechnology. Nat Biotech 28:1057–1068. doi: 10.1038/nbt.1685 CrossRefGoogle Scholar
  21. Qiu W, Baccarelli A, Carey VJ, Boutaoui N, Bacherman H, Klanderman B, Rennard S, Agusti A, Anderson W, Lomas DA, DeMeo DL (2012) Variable DNA methylation is associated with chronic obstructive pulmonary disease and lung function. Am J Respir Crit Care Med 185:373–381. doi: 10.1164/rccm.201108-1382OC CrossRefGoogle Scholar
  22. Robbins SLC, Ramzi S (2004) Pathological basis of disease. Saunders, PhiladelphiaGoogle Scholar
  23. Rosicarelli B, Stefanini S (2009) DEHP effects on histology and cell proliferation in lung of newborn rats. Histochem Cell Biol 131:491–500. doi: 10.1007/s00418-008-0550-4 CrossRefGoogle Scholar
  24. Sekaran S, Jagadeesan A (2015) In utero exposure to phthalate downregulates critical genes in Leydig cells of F1 male progeny. J Cell Biochem 116:1466–1477. doi: 10.1002/jcb.25108 CrossRefGoogle Scholar
  25. Stefanowicz D, Hackett TL, Garmaroudi FS, Günther OP, Neumann S, Sutanto EN, Ling KM, Kobor MS, Kicic A, Stick SM, Paré PD, Knight DA (2012) DNA methylation profiles of airway epithelial cells and PBMCs from healthy, atopic and asthmatic children. PLoS ONE 7:e44213. doi: 10.1371/journal.pone.0044213 CrossRefGoogle Scholar
  26. Tuomainen A, Stark H, Seuri M, Hirvonen MR, Linnainmaa M, Sieppi A, Tukiainen H (2006) Experimental PVC material challenge in subjects with occupational PVC exposure. Environ Health Perspect 114:1409–1413CrossRefGoogle Scholar
  27. Twentyman PR, Luscombe M (1987) A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Br J Cancer 56:279–285CrossRefGoogle Scholar
  28. Wormuth M, Scheringer M, Vollenweider M, Hungerbuhler K (2006) What are the sources of exposure to eight frequently used phthalic acid esters in Europeans? Risk Anal 26:803–824. doi: 10.1111/j.1539-6924.2006.00770.x CrossRefGoogle Scholar
  29. Zhang L, Sang H, Liu Y, Li J (2013) Manganese activates caspase-9-dependent apoptosis in human bronchial epithelial cells. Hum Exp Toxicol 32:1155–1163. doi: 10.1177/0960327112470272 CrossRefGoogle Scholar
  30. Zhao P, Zhong W, Ying X, Yuan Z, Fu J, Zhou Z (2008) Manganese chloride-induced G0/G1 and S phase arrest in A549 cells. Toxicology 250:39–46. doi: 10.1016/j.tox.2008.05.016 CrossRefGoogle Scholar
  31. Zhao P, Fu J, Yao B, Hu E, Song Y, Mi L, Li Z, Zhang H, Jia Y, Ma S, Chen W, Zhou Z (2013) Diethyl sulfate-induced cell cycle arrest and apoptosis in human bronchial epithelial 16HBE cells. Chem Biol Interact 205:81–89. doi: 10.1016/j.cbi.2013.06.014 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  • Yue Ma
    • 1
    • 2
  • Yinsheng Guo
    • 1
  • Shuang Wu
    • 1
  • Ziquan Lv
    • 1
  • Qian Zhang
    • 1
  • Xing Xie
    • 1
  • Yuebin Ke
    • 1
    Email author
  1. 1.Key Laboratory of Molecular BiologyShenzhen Center for Disease Control and PreventionShenzhenPeople’s Republic of China
  2. 2.Key Laboratory of Environment and Health (HUST), Ministry of Education & Ministry of Environmental Protection, and State Key Laboratory of Environmental Health (Incubation), School of Public Health, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanPeople’s Republic of China

Personalised recommendations