Skip to main content Accessibility help

Embryonic exposures to mono-2-ethylhexyl phthalate induce larval steatosis in zebrafish independent of Nrf2a signaling

  • Karilyn E. Sant (a1) (a2), Hadley M. Moreau (a2) (a3), Larissa M. Williams (a3), Haydee M. Jacobs (a2), Anna M. Bowsher (a3), Jason D. Boisvert (a3), Roxanna M. Smolowitz (a4), Jacob Pantazis (a3), Kate Annunziato (a2), Malina Nguyen (a2) and Alicia Timme-Laragy (a2)...


Mono-2-ethylhexyl phthalate (MEHP) is the primary metabolite of the ubiquitous plasticizer and toxicant, di-2-ethylhexyl phthalate. MEHP exposure has been linked to abnormal development, increased oxidative stress, and metabolic syndrome in vertebrates. Nuclear factor, Erythroid 2 Like 2 (Nrf2), is a transcription factor that regulates gene expression in response to oxidative stress. We investigated the role of Nrf2a in larval steatosis following embryonic exposure to MEHP. Wild-type and nrf2a mutant (m) zebrafish embryos were exposed to 0 or 200 μg/l MEHP from 6 to either 96 (histology) or 120 hours post fertilization (hpf). At 120 hpf, exposures were ceased and fish were maintained in clean conditions until 15 days post fertilization (dpf). At 15 dpf, fish lengths and lipid content were examined, and the expression of genes involved in the antioxidant response and lipid processing was quantified. At 96 hpf, a subset of animals treated with MEHP had vacuolization in the liver. At 15 dpf, deficient Nrf2a signaling attenuated fish length by 7.7%. MEHP exposure increased hepatic steatosis and increased expression of peroxisome proliferator-activated receptor alpha target fabp1a1. Cumulatively, these data indicate that developmental exposure alone to MEHP may increase risk for hepatic steatosis and that Nrf2a does not play a major role in this phenotype.


Corresponding author

Address for correspondence: Karilyn Sant, School of Public Health, San Diego State University, San Diego, CA92128, USA. Email:


Hide All
1.Phthalates Action Plan, U.S.E.P. Agency, Editor. 2012.
2.Benjamin, S, Masai, E, Kamimura, N, Takahashi, K, Anderson, RC, Faisal, PA. Phthalates impact human health: epidemiological evidences and plausible mechanism of action. J Hazard Mater. 2017; 340, 360383.
3.Wittassek, M, Angerer, J. Phthalates: metabolism and exposure. Int J Androl. 2008; 31, 131138.
4.Latini, G, De Felice, C, Verrotti, A. Plasticizers, infant nutrition and reproductive health. Reprod Toxicol. 2004; 19, 2733.
5.Frederiksen, H, Skakkebaek, NE, Andersson, AM. Metabolism of phthalates in humans. Mol Nutr Food Res. 2007; 51, 899911.
6.Inada, H, Chihara, K, Yamashita, A, et al.Evaluation of ovarian toxicity of mono-(2-ethylhexyl) phthalate (MEHP) using cultured rat ovarian follicles. J Toxicol Sci. 2012; 37, 483490.
7.Tomita, I, Nakamura, Y, Yagi, Y, Tutikawa, K. Fetotoxic effects of mono-2-ethylhexyl phthalate (MEHP) in mice. Environ Health Perspect. 1986; 65, 249254.
8.Guibert, E, Prieur, B, Cariou, R, et al.Effects of mono-(2-ethylhexyl) phthalate (MEHP) on chicken germ cells cultured in vitro. Environ Sci Pollut Res. 2013; 20, 27712783.
9.Jacobs, HM, Sant, KE, Basnet, A, Williams, LM, Moss, JB, Timme-Laragy, AR. Embryonic exposure to Mono(2-ethylhexyl) phthalate (MEHP) disrupts pancreatic organogenesis in zebrafish (Danio rerio). Chemosphere. 2018; 195, 498507.
10.Sant, KE, Dolinoy, DC, Jilek, JL, Sartor, MA, Harris, C. Mono-2-ethylhexyl phthalate disrupts neurulation and modifies the embryonic redox environment and gene expression. Reprod Toxicol. 2016; 63, 3248.
11.Swan, SH, Sathyanarayana, S, Barrett, ES, et al.First trimester phthalate exposure and anogenital distance in newborns. Human Reproduction (Oxford, England) 2015; 30, 963972.
12.Sathyanarayana, S, Barrett, E, Nguyen, R, Redmon, B, Haaland, W, Swan, SH. First trimester phthalate exposure and infant birth weight in the infant development and environment study. Int J Environ Res Public Health 2016; 13, 945.
13.Harley, KGBerger, K, Rauch, S, et al. Association of prenatal urinary phthalate metabolite concentrations and childhood BMI and obesity. Pediatr Res. 2017; 82, 405415.
14.Heindel, JJ. History of the obesogen field: looking back to look forward. Front Endocrinol. 2019; 10, 1414.
15.Heindel, JJ, Blumberg, B. Environmental obesogens: mechanisms and controversies. Annu Rev Pharmacol Toxicol. 2019; 59, 89106.
16.Grün, F, Blumberg, B. Endocrine disrupters as obesogens. Mol Cell Endocrinol. 2009; 304, 1929.
17.Grün, F, Blumberg, B. Environmental obesogens: organotins and endocrine disruption via nuclear receptor signaling. Endocrinology. 2006; 147, s50s55.
18.Wang, W, Craig, ZR, Basavarajappa, MS, Hafner, KS, Flaws, JA. Mono-(2-ethylhexyl) phthalate induces oxidative stress and inhibits growth of mouse ovarian antral follicles. Biol Reprod. 2012; 87, 152152.
19.Pi, J, Leung, L, Xue, P, et al.Deficiency in the nuclear factor E2-related Factor-2 transcription factor results in impaired adipogenesis and protects against diet-induced obesity. J Biol Chem. 2010; 285, 92929300.
20.Sheikh, IA, Abu-Elmagd, M, Turki, RF, Damanhouri, GA, Beg, MA, Al-Qahtani, M. Endocrine disruption: in silico perspectives of interactions of di-(2-ethylhexyl)phthalate and its five major metabolites with progesterone receptor. BMC Struct Biol. 2016; 16(Suppl. 1), 1616.
21.Zhai, W, Huang, Z, Chen, L, Feng, C, Li, B, Li, T. Thyroid endocrine disruption in zebrafish larvae after exposure to mono-(2-Ethylhexyl) phthalate (MEHP). PLoS One. 2014; 9, e92465.
22.Shelton, P, Jaiswal, AK. The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene? FASEB J. 2013; 27, 414423.
23.Kwong, M, Kan, YW, Chan, JY. The CNC basic leucine zipper factor, Nrf1, is essential for cell survival in response to oxidative stress-inducing agents. Role for Nrf1 in gamma-gcs(l) and gss expression in mouse fibroblasts. J Biol Chem. 2000; 274; 3749137498.
24.Sykiotis, GP, Bohmann, D. Stress-activated cap’n’collar transcription factors in aging and human disease. Sci Signal. 2010; 3, re3re3.
25.Ma, Q. Role of nrf2 in oxidative stress and toxicity. Annu Rev Pharmacol Toxicol. 2013; 53, 401426.
26.Espinosa-Diez, C, Miguel, V, Mennerich, D, et al.Antioxidant responses and cellular adjustments to oxidative stress. Redox Biol. 2015; 6, 183197.
27.Timme-Laragy, AR, Karchner, SI, Franks, DG, et al.Nrf2b, novel zebrafish paralog of oxidant-responsive transcription factor NF-E2-related factor 2 (NRF2). J Biol Chem. 2012; 287, 46094627.
28.Kobayashi, M, Itoh, K, Suzuki, T, et al.Identification of the interactive interface and phylogenic conservation of the Nrf2-Keap1 system. Genes Cells. 2002; 7, 807820.
29.Sant, KE, et al.Nrf2a modulates the embryonic antioxidant response to perfluorooctanesulfonic acid (PFOS) in the zebrafish, Danio rerio. Aquat Toxicol. 2018; 198, 92102.
30.Sant, KE, et al. The role of Nrf1 and Nrf2 in the regulation of glutathione and redox dynamics in the developing zebrafish embryo. Redox Biol. 2017; 13 (Suppl. C), 207218.
31.Furukawa, S, Fujita, T, Shimabukuro, M, et al.Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest. 2004; 114, 17521761.
32.Marseglia, L, Manti, S, D’Angelo, G, et al.Oxidative stress in obesity: a critical component in human diseases. Int J Mol Sci. 2014; 16, 378400.
33.Takahashi, T, et al.Carnosic acid and carnosol inhibit adipocyte differentiation in mouse 3T3-L1 cells through induction of phase2 enzymes and activation of glutathione metabolism. Biochem Biophys Res Commun. 2009; 382, 549554.
34.Shin, S, Wakabayashi, J, Yates, MS, et al.Role of Nrf2 in prevention of high-fat diet-induced obesity by synthetic triterpenoid CDDO-Imidazolide. Eur J Pharmacol. 2009; 620, 138144.
35.Seo, H-A, Lee, I-K. The role of Nrf2: adipocyte differentiation, obesity, and insulin resistance. Oxid Med Cell Longevity. 2013; 2013, 184598184598.
36.Physiology, Liver. 2018 18 December 2018 [cited 4 March 2019]; Available from:
37.Benedict, M, Zhang, X. Non-alcoholic fatty liver disease: an expanded review. World J Hepatol. 2017; 9, 715732.
38.Sayiner, M, et al., Epidemiology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in the United States and the rest of the world. Clin Liver Dis. 2016; 20, 205214.
39.Arciello, M, Gori, M, Maggio, R, et al.Environmental pollution: a tangible risk for NAFLD pathogenesis. Int J Mol Sci. 2013; 14.
40.Mukaigasa, K, et al.Genetic evidence of an evolutionarily conserved role for Nrf2 in the protection against oxidative stress. Mol Cell Biol. 2012; 32, 44554461.
41.Rousseau, ME, et al.Regulation of Ahr signaling by Nrf2 during development: effects of Nrf2a deficiency on PCB126 embryotoxicity in zebrafish (Danio rerio). Aquat Toxicol. (Amsterdam, Netherlands) 2015; 167, 157171.
42.Schlegel, A, Stainier, DYR. Microsomal triglyceride transfer protein is required for yolk lipid utilization and absorption of dietary lipids in zebrafish larvae. Biochemistry. 2006; 45, 1517915187.
43.Fraher, D, et al. Lipid abundance in zebrafish embryos is regulated by complementary actions of the endocannabinoid system and retinoic acid pathway. Endocrinology. 2015; 156, 35963609.
44.Livak, KJ, Schmittgen, TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001; 25, 402408.
45.McCurley, AT, Callard, GVJBMB. Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment. BMC Mol Biol. 2008; 9, 102.
46.Cooper, CA, Handy, RD, Bury, NR. The effects of dietary iron concentration on gastrointestinal and branchial assimilation of both iron and cadmium in zebrafish (Danio rerio). Aquat Toxicol. 2006; 79, 167175.
47.Zhao, X, et al.Klf6/copeb is required for hepatic outgrowth in zebrafish and for hepatocyte specification in mouse ES cells. Dev Biol. 2010; 344, 7993.
48.Laprairie, RB, Denovan-Wright, EM, Wright, JM. Subfunctionalization of peroxisome proliferator response elements accounts for retention of duplicated fabp1 genes in zebrafish. BMC Evol Biol. 2016; 16, 147.
49.Schultz, LE, et al.Epigenetic regulators Rbbp4 and Hdac1 are overexpressed in a zebrafish model of RB1 embryonal brain tumor, and are required for neural progenitor survival and proliferation. Dis Model Mech. 2018; 11, dmm034124.
50.Williams, LM, et al.Developmental expression of the Nfe2-related factor (Nrf) transcription factor family in the Zebrafish, Danio rerio. PLoS One. 2013; 8, e79574.
51.Williams, LM, et al.The transcription factor, Nuclear factor, erythroid 2 (Nfe2), is a regulator of the oxidative stress response during Danio rerio development. Aquat Toxicol. (Amsterdam, Netherlands) 2016; 180, 141154.
52.Cederbaum, AI, Nrf2 and antioxidant defense against CYP2E1 toxicity. In Cytochrome P450 2E1: Its Role in Disease and Drug Metabolism (ed. Dey, A), 2013; pp. 105130. Springer Netherlands, Dordrecht.
53.Thisse, B, et al. Expression of the zebrafish genome during embryogenesis (NIH R01 RR15402), in ZFIN Direct Data Submission ( 2001.
54.Fagerberg, L, et al.Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics. 2014; 13, 397406.
55.Feige, JN, et al.The endocrine disruptor monoethyl-hexyl-phthalate is a selective peroxisome proliferator-activated receptor γ modulator that promotes adipogenesis. J Biol Chem. 2007; 282, 1915219166.
56.Hurst, CH, Waxman, DJ. Activation of PPARα and PPARγ by environmental phthalate monoesters. Toxicol Sci. 2003; 74, 297308.
57.Sant, KE, et al.Embryonic exposures to perfluorooctanesulfonic acid (PFOS) disrupt pancreatic organogenesis in the zebrafish, Danio rerio. Environ Pollut. 2017; 220, 807817.
58.Fenton, TR, Kim, JH. A systematic review and meta-analysis to revise the Fenton growth chart for preterm infants. BMC Pediatr. 2013; 13, 59.
59.Dulloo, AG, et al.The thrifty ‘catch-up fat’ phenotype: its impact on insulin sensitivity during growth trajectories to obesity and metabolic syndrome. Int J Obes. 2006; 30, S23S35.
60.Vaag, A. Low birth weight and early weight gain in the metabolic syndrome: consequences for infant nutrition. Int J Gynecol Obstet. 2009; 104 (Supplement), S32S34.
61.Simmons, R. Developmental origins of adult metabolic disease: concepts and controversies. Trends Endocrinol Metabol. 2005; 16, 390394.
62.Pan, L, et al.Trends in severe obesity among children aged 2 to 4 years enrolled in special supplemental nutrition program for women, infants, and children from 2000 to 2014 trends in severe obesity among US children aged 2 to 4 years enrolled in WICTrends in severe obesity among US children aged 2 to 4 years enrolled in WIC. JAMA Pediatr. 2018; 172, 232238.
63.Fryar, CD, Carroll, MD, Ogden, CL. Prevalence of Overweight and Obesity among Children and Adolescents: United States, 1963–1965 Through 2011–2012, N.C.f.H. Statistics, Editor. 2014, Centers for Disease Control and Prevention, Division of Health and Nutrition Examination Surveys. Hyattsville, MD.
64.Bai, J, et al.Mono-2-ethylhexyl phthalate induces the expression of genes involved in fatty acid synthesis in HepG2 cells. Environ Toxicol Pharmacol. 2019; 69, 104111.
65.Zhang, Y, et al.Mono-2-ethylhexyl phthalate (MEHP) promoted lipid accumulation via JAK2/STAT5 and aggravated oxidative stress in BRL-3A cells. Ecotoxicol Environ Saf. 2019; 184, 109611.
66.Pereira, C, Mapuskar, K, Rao, CV. Chronic toxicity of diethyl phthalate in male Wistar rats – a dose–response study. Regul Toxicol Pharm. 2006; 45, 169177.
67.Ibabe, A, Bilbao, E, Cajaraville, MP. Expression of peroxisome proliferator-activated receptors in zebrafish (Danio rerio) depending on gender and developmental stage. Histochem Cell Biol. 2005; 123, 7587.
68.Liss, KHH, Finck, BN. PPARs and nonalcoholic fatty liver disease. Biochimie. 2017; 136, 6574.
69.Liew, WC, et al.Polygenic sex determination system in zebrafish. PLoS One. 2012; 7, e34397e34397.
70.Uchida, D, et al.Oocyte apoptosis during the transition from ovary-like tissue to testes during sex differentiation of juvenile zebrafish. J Exp Biol. 2002; 205, 711718.
71.Phillips, ML. Phthalates and metabolism: exposure correlates with obesity and diabetes in men. Environ Health Perspect. 2007; 115, A312A312.
72.Ballestri, S, et al.NAFLD as a sexual dimorphic disease: role of gender and reproductive status in the development and progression of nonalcoholic fatty liver disease and inherent cardiovascular risk. Adv Therapy. 2017; 34, 12911326.
73.Bertolotti, M, et al.Nonalcoholic fatty liver disease and aging: epidemiology to management. World J Gastroenterol. 2014; 20, 1418514204.


Type Description Title
Supplementary materials

Sant et al. supplementary material
Sant et al. supplementary material 1

 Unknown (963 KB)
963 KB
Supplementary materials

Sant et al. supplementary material
Sant et al. supplementary material 2

 Unknown (730 KB)
730 KB
Supplementary materials

Sant et al. supplementary material
Sant et al. supplementary material 3

 Word (28 KB)
28 KB

Embryonic exposures to mono-2-ethylhexyl phthalate induce larval steatosis in zebrafish independent of Nrf2a signaling

  • Karilyn E. Sant (a1) (a2), Hadley M. Moreau (a2) (a3), Larissa M. Williams (a3), Haydee M. Jacobs (a2), Anna M. Bowsher (a3), Jason D. Boisvert (a3), Roxanna M. Smolowitz (a4), Jacob Pantazis (a3), Kate Annunziato (a2), Malina Nguyen (a2) and Alicia Timme-Laragy (a2)...


Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed.