Environmental Science and Pollution Research

, Volume 24, Issue 20, pp 16659–16672 | Cite as

Urinary metabolomic profiling in rats exposed to dietary di(2-ethylhexyl) phthalate (DEHP) using ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS)

  • Xinwen Dong
  • Yunbo Zhang
  • Jin Dong
  • Yue Zhao
  • Jipeng Guo
  • Zhanju Wang
  • Mingqi Liu
  • Xiaolin Na
  • Cheng Wang
Research Article


Di(2-ethylhexyl) phthalate (DEHP) is an omnipresent environmental chemical with widespread nonoccupational human exposure through multiple ways. Although considerable efforts have been invested to investigate mechanisms of DEHP toxicity, the key metabolic biomarkers of DEHP toxicity remain to be identified. The aim of this study was to assess the urinary metabonomics of dietary DEHP in rats using the technique of ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS). Fourteen female Wistar rats were divided into two groups and given increasing dietary doses of DEHP for 30 consecutive days. The urinary metabolite profile was studied using ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) enabled clusters to be clearly separated. Eleven principal urinary metabolites were identified as contributing to the clusters. The clusters in the positive electrospray ionization (ESI) mode were xanthurenic acid, kynurenic acid, nonate, N6-methyladenosine, and L-isoleucyl-L-proline. The clusters in the negative ESI mode were hippuric acid, tetrahydrocortisol, citric acid, phenylpropionylglycine, cPA(18:2(9Z, 12Z)/0:0), and LysoPC(14:1(9Z)). The urinary metabonomic changes indicated that exposure to dietary DEHP can affect energy-related metabolism, liver and renal function, fatty acid metabolism, and cause DNA damage in rats. The findings of this study on the urinary metabolites and metabolic pathways of DEHP may form the basis for future studies on the mechanisms of toxicity of this commonly found environmental chemical.


Di(2-ethylhexyl) phthalate Environmental chemical Toxic effects Metabonomics Biomarker Mechanism 



This work was supported by the National Natural Science Foundation of China (Grant No. 81273079). The present study was conducted in the laboratory of the Key Laboratory of Nutrition and Food Hygiene (Harbin Medical University), Heilongjiang Higher Education Institutions.

Compliance with ethical standards

All animal care and experimental procedures were approved by the Committee on the Ethics of Animal Experiments of the University of Harbin Medical and were in accordance with the policies on the care and use of animals of the current Chinese legislation.

Conflict of interest

The authors declare that they have no conflict of interest.

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  1. Ait Bamai Y et al (2014) Exposure to house dust phthalates in relation to asthma and allergies in both children and adults. Sci Total Environ 485-486:153–163. doi: 10.1016/j.scitotenv.2014.03.059 CrossRefGoogle Scholar
  2. Barr DB et al (2003) Assessing human exposure to phthalates using monoesters and their oxidized metabolites as biomarkers. Environ Health Perspect 111:1148–1151. doi: 10.1289/ehp.6074 CrossRefGoogle Scholar
  3. Bridges JW, French MR, Smith RL, Williams RT (1970) The fate of benzoic acid in various species. Biochem J 118:47–51CrossRefGoogle Scholar
  4. Briere JJ, Favier J, El Ghouzzi V, Djouadi F, Benit P, Gimenez AP, Rustin P (2005) Succinate dehydrogenase deficiency in human. Cell Mol Life Sci 62:2317–2324. doi: 10.1007/s00018-005-5237-6 CrossRefGoogle Scholar
  5. Chang JW, Lee CC, Pan WH, Chou WC, Huang HB, Chiang HC, Huang PC (2017) Estimated daily intake and cumulative risk assessment of phthalates in the General Taiwanese after the 2011 DEHP Food Scandal. Sci Rep 7:45009. doi: 10.1038/srep45009 CrossRefGoogle Scholar
  6. Chen H, Zhang W, Rui BB, Yang SM, Xu WP, Wei W (2016) Di(2-ethylhexyl) phthalate exacerbates non-alcoholic fatty liver in rats and its potential mechanisms. Environ Toxicol Pharmacol 42:38–44. doi: 10.1016/j.etap.2015.12.016 CrossRefGoogle Scholar
  7. Chen ML, Chen JS, Tang CL, Mao IF (2008) The internal exposure of Taiwanese to phthalate—an evidence of intensive use of plastic materials. Environ Int 34:79–85. doi: 10.1016/j.envint.2007.07.004 CrossRefGoogle Scholar
  8. Cho YJ, Park SB, Han M (2015) Di-(2-ethylhexyl)-phthalate induces oxidative stress in human endometrial stromal cells in vitro. Mol Cell Endocrinol 407:9–17. doi: 10.1016/j.mce.2015.03.003 CrossRefGoogle Scholar
  9. Chun J et al (2002) International union of pharmacology. XXXIV. Lysophospholipid receptor nomenclature. Pharmacol Rev 54:265–269CrossRefGoogle Scholar
  10. David RM, Moore MR, Finney DC, Guest D (2000) Chronic toxicity of di (2-ethylhexyl)phthalate in rats. Toxicol Sci Off J Soc Toxicol 55:433–443CrossRefGoogle Scholar
  11. Dong X et al (2017) Effects of long-term in vivo exposure to di-2-ethylhexylphthalate on thyroid hormones and the TSH/TSHR signaling pathways in Wistar rats. Int J Environ Res Public Health 14(1):44. doi: 10.3390/ijerph14010044
  12. Duty SM et al (2003) The relationship between environmental exposures to phthalates and DNA damage in human sperm using the neutral comet assay. Environ Health Perspect 111:1164–1169CrossRefGoogle Scholar
  13. Euling SY, Thompson CM, Chiu WA, Benson R (2013) An approach for integrating toxicogenomic data in risk assessment: the dibutyl phthalate case study. Toxicol Appl Pharmacol 271:324–335. doi: 10.1016/j.taap.2013.03.013 CrossRefGoogle Scholar
  14. Fay M, Donohue JM, De Rosa C (1999) ATSDR evaluation of health effects of chemicals. VI. Di (2-ethylhexyl) phthalate. Agency Toxic Subst Dis Registry Toxicol Ind Health 15:651–746CrossRefGoogle Scholar
  15. Fisher JS, Macpherson S, Marchetti N, Sharpe RM (2003) Human ‘testicular dysgenesis syndrome’: a possible model using in-utero exposure of the rat to dibutyl phthalate. Hum Reprod 18:1383–1394CrossRefGoogle Scholar
  16. Fromme H et al (2007) Intake of phthalates and di (2-ethylhexyl) adipate: results of the Integrated Exposure Assessment Survey based on duplicate diet samples and biomonitoring data. Environ Int 33:1012–1020. doi: 10.1016/j.envint.2007.05.006 CrossRefGoogle Scholar
  17. Fukuwatari T, Ohta M, Sugimoto E, Sasaki R, Shibata K (2004) Effects of dietary di (2-ethylhexyl) phthalate, a putative endocrine disrupter, on enzyme activities involved in the metabolism of tryptophan to niacin in rats. Biochimica et Biophysica Acta 1672:67–75. doi: 10.1016/j.bbagen.2004.02.009 CrossRefGoogle Scholar
  18. Gao H et al (2017) Cumulative risk assessment of phthalates associated with birth outcomes in pregnant Chinese women: a prospective cohort study. Environ Pollut 222:549–556. doi: 10.1016/j.envpol.2016.11.026 CrossRefGoogle Scholar
  19. Gaspar FW, Castorina R, Maddalena RL, Nishioka MG, McKone TE, Bradman A (2014) Phthalate exposure and risk assessment in California child care facilities. Environ Sci Technol 48:7593–7601. doi: 10.1021/es501189t CrossRefGoogle Scholar
  20. Gray LE Jr, Ostby J, Furr J, Price M, Veeramachaneni DN, Parks L (2000) Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat. Toxicol Sci 58:350–365CrossRefGoogle Scholar
  21. Halden RU (2010) Plastics and health risks. Annu Rev Public Health 31:179–194. doi: 10.1146/annurev.publhealth.012809.103714 CrossRefGoogle Scholar
  22. Hannas BR, Lambright CS, Furr J, Howdeshell KL, Wilson VS, Gray LE Jr (2011) Dose-response assessment of fetal testosterone production and gene expression levels in rat testes following in utero exposure to diethylhexyl phthalate, diisobutyl phthalate, diisoheptyl phthalate, and diisononyl phthalate. Toxicol Sci Off J Soc Toxicol 123:206–216. doi: 10.1093/toxsci/kfr146 CrossRefGoogle Scholar
  23. Hokanson R, Hanneman W, Hennessey M, Donnelly KC, McDonald T, Chowdhary R, Busbee DL (2006) DEHP, bis (2)-ethylhexyl phthalate, alters gene expression in human cells: possible correlation with initiation of fetal developmental abnormalities. Hum Exp Toxicol 25:687–695CrossRefGoogle Scholar
  24. Howdeshell KL, Hotchkiss AK, Gray LE Jr (2016) Cumulative effects of antiandrogenic chemical mixtures and their relevance to human health risk assessment. Int J Hygiene Environ Health 220(2 Pt A):179–188. doi: 10.1016/j.ijheh.2016.11.007
  25. Hsu PC et al (2016) The adverse effects of low-dose exposure to di (2-ethylhexyl) phthalate during adolescence on sperm function in adult rats. Environ Toxicol 31:706–712. doi: 10.1002/tox.22083 CrossRefGoogle Scholar
  26. Hu X et al (2014) 11beta-hydroxysteroid dehydrogenase type 2 enzyme activity effect after exposures phthalate esters in maternal. Zhonghua yu fang yi xue za zhi [Chin J Prev Med] 48:800–804Google Scholar
  27. Huang Q, Zhang H, Chen YJ, Chi YL, Dong S (2016) The inflammation response to DEHP through PPARgamma in endometrial cells. Int J Environ Res Public Health 13(3):318. doi: 10.3390/ijerph13030318
  28. Iwanowicz LR et al (2016) Evidence of estrogenic endocrine disruption in smallmouth and largemouth bass inhabiting Northeast U.S. national wildlife refuge waters: a reconnaissance study. Ecotoxicol Environ Safety 124:50–59. doi: 10.1016/j.ecoenv.2015.09.035 CrossRefGoogle Scholar
  29. Jia G et al (2011) N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol 7:885–887. doi: 10.1038/nchembio.687 CrossRefGoogle Scholar
  30. Jurewicz J, Hanke W (2011) Exposure to phthalates: reproductive outcome and children health. A review of epidemiological studies. Int J Occup Med Environ Health 24:115–141. doi: 10.2478/s13382-011-0022-2 CrossRefGoogle Scholar
  31. Kato K et al (2003) Mono (2-ethyl-5-hydroxyhexyl) phthalate and mono-(2-ethyl-5-oxohexyl) phthalate as biomarkers for human exposure assessment to di-(2-ethylhexyl) phthalate. Environ Health Perspect 112:327–330. doi: 10.1289/ehp.6663 CrossRefGoogle Scholar
  32. Kavlock R et al (2002) NTP Center for the Evaluation of Risks to Human Reproduction: phthalates expert panel report on the reproductive and developmental toxicity of di(2-ethylhexyl) phthalate. Reprod Toxicol 16:529–653CrossRefGoogle Scholar
  33. Kekkonen R-A (2008) Effect of probiotic Lactobacillus rhamnosus GG intervention on global serum lipidomic profiles in healthy adults. World J Gastroenterol 14:3188. doi: 10.3748/wjg.14.3188 CrossRefGoogle Scholar
  34. Klimisch HJ, Gamer AO, Hellwig J, Kaufmann W, Jackh R (1992) Di-(2-ethylhexyl) phthalate: a short-term repeated inhalation toxicity study including fertility assessment. Food Chem Toxicol 30:915–919CrossRefGoogle Scholar
  35. Kobayashi T, Tanaka-Ishii R, Taguchi R, Ikezawa H, Murakami-Murofushi K (1999) Existence of a bioactive lipid, cyclic phosphatidic acid, bound to human serum albumin. Life Sci 65:2185–2191CrossRefGoogle Scholar
  36. Koch HM, Rossbach B, Drexler H, Angerer J (2003) Internal exposure of the general population to DEHP and other phthalates—determination of secondary and primary phthalate monoester metabolites in urine. Environ Res 93:177–185CrossRefGoogle Scholar
  37. Lake BG, Gray TJ, Gangolli SD (1986) Hepatic effects of phthalate esters and related compounds—in vivo and in vitro correlations. Environ Health Perspect 67:283–290Google Scholar
  38. Lenz EM, Wilson ID (2007) Analytical strategies in metabonomics. J Proteome Res 6:443–458. doi: 10.1021/pr0605217 CrossRefGoogle Scholar
  39. Lien YJ et al (2015) Prenatal exposure to phthalate esters and behavioral syndromes in children at 8 years of age: Taiwan Maternal and Infant Cohort Study. Environ Health Perspect 123:95–100. doi: 10.1289/ehp.1307154 CrossRefGoogle Scholar
  40. Martinelli MI, Mocchiutti NO, Bernal CA (2010) Effect of di (2-ethylhexyl) phthalate (DEHP) on lipolysis and lipoprotein lipase activities in adipose tissue of rats. Hum Exp Toxicol 29:739–745. doi: 10.1177/0960327110361750 CrossRefGoogle Scholar
  41. Mendiola J et al (2012) Urinary concentrations of di (2-ethylhexyl) phthalate metabolites and serum reproductive hormones: pooled analysis of fertile and infertile men. J Androl 33:488–498. doi: 10.2164/jandrol.111.013557 CrossRefGoogle Scholar
  42. Miao Y, Wang R, Lu C, Zhao J, Deng Q (2017) Lifetime cancer risk assessment for inhalation exposure to di (2-ethylhexyl) phthalate (DEHP). Environ Sci Pollut Res Int 24:312–320. doi: 10.1007/s11356-016-7797-4 CrossRefGoogle Scholar
  43. Murakami-Murofushi K et al (1995) Selective inhibition of DNA polymerase-alpha family with chemically synthesized derivatives of PHYLPA, a unique Physarum lysophosphatidic acid. Biochim Biophys Acta 1258:57–60CrossRefGoogle Scholar
  44. National Research Council (2008) Phthalates and cumulative risk assessment the task ahead. The National Academies Press, Washington, DC. doi: 10.17226/12528
  45. Nicholson JK, Lindon JC, Holmes E (1999) ‘Metabonomics’: understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica 29:1181–1189. doi: 10.1080/004982599238047 CrossRefGoogle Scholar
  46. Niu J, Pi Z, Yue H, Wang Y, Yu Q, Liu S (2012) Effect of ginseng polysaccharide on the urinary excretion of type 2 diabetic rats studied by liquid chromatography-mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 907:7–12. doi: 10.1016/j.jchromb.2012.08.012 CrossRefGoogle Scholar
  47. Pak VM, McCauley LA, Pinto-Martin J (2011) Phthalate exposures and human health concerns: a review and implications for practice. AAOHN J 59:228–233; quiz 234-225. doi: 10.3928/08910162-20110426-01 CrossRefGoogle Scholar
  48. Pan T (2013) N6-methyl-adenosine modification in messenger and long non-coding RNA. Trends Biochem Sci 38:204–209. doi: 10.1016/j.tibs.2012.12.006 CrossRefGoogle Scholar
  49. Pawlak D, Tankiewicz A, Buczko W (2001) Kynurenine and its metabolites in the rat with experimental renal insufficiency. J Physiol Pharmacol Off J Pol Physiol Soc 52:755–766Google Scholar
  50. Petersen JH, Breindahl T (2000) Plasticizers in total diet samples, baby food and infant formulae. Food Addit Contam 17:133–141. doi: 10.1080/026520300283487 CrossRefGoogle Scholar
  51. Rask E, Simonyte K, Lonn L, Axelson M (2013) Cortisol metabolism after weight loss: associations with 11 beta-HSD type 1 and markers of obesity in women. Clin Endocrinol 78:700–705. doi: 10.1111/j.1365-2265.2012.04333.x CrossRefGoogle Scholar
  52. Schettler T (2006) Human exposure to phthalates via consumer products. Int J Androl 29:134–139; discussion 181-135. doi: 10.1111/j.1365-2605.2005.00567.x CrossRefGoogle Scholar
  53. Scott AP, Ellis T, Tveiten H (2014) Identification of cortisol metabolites in the bile of Atlantic cod Gadus morhua L. Steroids 88:26–35. doi: 10.1016/j.steroids.2014.05.014 CrossRefGoogle Scholar
  54. Serrano SE, Braun J, Trasande L, Dills R, Sathyanarayana S (2014) Phthalates and diet: a review of the food monitoring and epidemiology data. Environ Health 13:43. doi: 10.1186/1476-069X-13-43 CrossRefGoogle Scholar
  55. Sharpe RM (2001) Hormones and testis development and the possible adverse effects of environmental chemicals. Toxicol Lett 120:221–232CrossRefGoogle Scholar
  56. Skakkebaek NE (2002) Endocrine disrupters and testicular dysgenesis syndrome. Horm Res 57(Suppl 2):43Google Scholar
  57. Strakovsky RS, Lezmi S, Shkoda I, Flaws JA, Helferich WG, Pan YX (2015) In utero growth restriction and catch-up adipogenesis after developmental di (2-ethylhexyl) phthalate exposure cause glucose intolerance in adult male rats following a high-fat dietary challenge. J Nutr Biochem 26:1208–1220. doi: 10.1016/j.jnutbio.2015.05.012 CrossRefGoogle Scholar
  58. Sun X, Xu W, Zeng Y, Hou Y, Guo L, Zhao X, Sun C (2014) Metabonomics evaluation of urine from rats administered with phorate under long-term and low-level exposure by ultra-performance liquid chromatography-mass spectrometry. J Appl Toxicol 34:176–183. doi: 10.1002/jat.2848 CrossRefGoogle Scholar
  59. Trygg J, Holmes E, Lundstedt T (2007) Chemometrics in metabonomics. J Proteome Res 6:469–479. doi: 10.1021/pr060594q CrossRefGoogle Scholar
  60. Wang X et al (2015) Oxidative DNA damage induced by di-(2-ethylhexyl) phthalate in HEK-293 cell line. Environ Toxicol Pharmacol 39:1099–1106. doi: 10.1016/j.etap.2015.03.016 CrossRefGoogle Scholar
  61. Wirth JJ et al (2008) A pilot study associating urinary concentrations of phthalate metabolites and semen quality. Syst Biol Reprod Med 54:143–154. doi: 10.1080/19396360802055921 CrossRefGoogle Scholar
  62. Yetukuri L, Katajamaa M, Medina-Gomez G, Seppanen-Laakso T, Vidal-Puig A, Oresic M (2007) Bioinformatics strategies for lipidomics analysis: characterization of obesity related hepatic steatosis. BMC Syst Biol 1:12. doi: 10.1186/1752-0509-1-12 CrossRefGoogle Scholar
  63. Zhang C, Zhang M, Sun Y, Li J, Fang M, Zhu X, Liu C (2012) Effect of dibutyl phthalate and di-(2-ethylhexyl) phthalate on urine SOD activity and MDA content in rats. Nan fang yi ke da xue xue bao = J Southern Med Univ 32:160–164Google Scholar
  64. Zhao X, Yang Y, Sun BF, Zhao YL, Yang YG (2014) FTO and obesity: mechanisms of association. Curr Diab Rep 14:486. doi: 10.1007/s11892-014-0486-0 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Xinwen Dong
    • 1
  • Yunbo Zhang
    • 1
  • Jin Dong
    • 1
  • Yue Zhao
    • 1
  • Jipeng Guo
    • 1
  • Zhanju Wang
    • 1
  • Mingqi Liu
    • 1
  • Xiaolin Na
    • 1
  • Cheng Wang
    • 1
  1. 1.Department of Environmental Hygiene, Public Health CollegeHarbin Medical UniversityHarbinChina

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