Research Article

Alteration of the N⁶-methyladenosine epitranscriptomic profile in synthetic phthalate-treated human induced pluripotent stem cell-derived endothelial cells

Department of Pharmacology & Regenerative Medicine, The University of Illinois College of Medicine, 909 S Wolcott Ave, COMRB 4100, Chicago, IL 60612, USA

‡Co-first authors

Search for more papers by this author

Zhenbo Han‡

https://orcid.org/0000-0001-6892-5163

Department of Pharmacology & Regenerative Medicine, The University of Illinois College of Medicine, 909 S Wolcott Ave, COMRB 4100, Chicago, IL 60612, USA

‡Co-first authors

Search for more papers by this author

Gege Yan‡

https://orcid.org/0000-0001-5923-4862

Department of Pharmacology & Regenerative Medicine, The University of Illinois College of Medicine, 909 S Wolcott Ave, COMRB 4100, Chicago, IL 60612, USA

‡Co-first authors

Search for more papers by this author

Sarath Babu Nukala

https://orcid.org/0000-0002-0216-7168

Department of Pharmacology & Regenerative Medicine, The University of Illinois College of Medicine, 909 S Wolcott Ave, COMRB 4100, Chicago, IL 60612, USA

Search for more papers by this author

Youjeong Kwon

https://orcid.org/0000-0002-3915-8740

Department of Pharmacology & Regenerative Medicine, The University of Illinois College of Medicine, 909 S Wolcott Ave, COMRB 4100, Chicago, IL 60612, USA

Search for more papers by this author

Hoai Huong Thi Le

https://orcid.org/0000-0003-1843-2349

Department of Basic Medical Sciences, University of Arizona College of Medicine, ABC-1 Building, 425 North 5th Street, Phoenix, AZ 85004, USA

Search for more papers by this author

Ya Li

Department of Pharmacology & Regenerative Medicine, The University of Illinois College of Medicine, 909 S Wolcott Ave, COMRB 4100, Chicago, IL 60612, USA

Search for more papers by this author

Sang-Bing Ong

https://orcid.org/0000-0001-7421-2610

Department of Medicine & Therapeutics, Faculty of Medicine, Chinese University of Hong Kong (CUHK), Hong Kong SAR, China

Centre for Cardiovascular Genomics & Medicine (CCGM), Lui Che Woo Institute of Innovative Medicine, CUHK, Hong Kong SAR, China

Hong Kong Hub of Paediatric Excellence (HK HOPE), Hong Kong Children's Hospital (HKCH), Kowloon Bay, Hong Kong SAR, China

Kunming Institute of Zoology – The Chinese University of Hong Kong (KIZ-CUHK) Joint Laboratory of Bioresources & Molecular Research of Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223 Yunnan, China

Search for more papers by this author

Won Hee Lee

*Author for correspondence: Tel.: +1 602 827 2142;

E-mail Address: whlee@arizona.edu

https://orcid.org/0000-0001-8534-9639

Department of Basic Medical Sciences, University of Arizona College of Medicine, ABC-1 Building, 425 North 5th Street, Phoenix, AZ 85004, USA

Search for more papers by this author

Sang-Ging Ong

**Author for correspondence: Tel.: +1 312 996 7665;

E-mail Address: sangging@uic.edu

https://orcid.org/0000-0003-0182-8769

Department of Pharmacology & Regenerative Medicine, The University of Illinois College of Medicine, 909 S Wolcott Ave, COMRB 4100, Chicago, IL 60612, USA

Department of Medicine & Therapeutics, Faculty of Medicine, Chinese University of Hong Kong (CUHK), Hong Kong SAR, China

Division of Cardiology, Department of Medicine, The University of Illinois College of Medicine, 909 S Wolcott Ave, COMRB 4100, Chicago, IL 60612, USA

Search for more papers by this author

Published Online:31 Oct 2022https://doi.org/10.2217/epi-2022-0110

View article

Abstract

Background: This study aimed to characterize the N⁶-methyladenosine epitranscriptomic profile induced by mono(2-ethylhexyl) phthalate (MEHP) exposure using a human-induced pluripotent stem cell-derived endothelial cell model. Methods: A multiomic approach was employed by performing RNA sequencing in parallel with an N⁶-methyladenosine-specific microarray to identify mRNAs, lncRNAs, and miRNAs affected by MEHP exposure. Results: An integrative multiomic analysis identified relevant biological features affected by MEHP, while functional assays provided a phenotypic characterization of these effects. Transcripts regulated by the epitranscriptome were validated with quantitative PCR and methylated RNA immunoprecipitation. Conclusion: The authors' profiling of the epitranscriptome expands the scope of toxicological insights into known environmental toxins to under surveyed cellular contexts and emerging domains of regulation and is, therefore, a valuable resource to human health.

Plain language summary

Synthetic phthalates, such as mono(2-ethyhexyl) phthalate, have long been recognized as environmental toxins. What effect these compounds have on endothelial cells remains poorly understood. To address this, the authors utilized a human-induced pluripotent stem cell-derived endothelial cell model to screen for an environmental toxin. They then obtained a profile of the epitranscriptomic changes involving the N⁶-methyladensosine modification and performed biochemical and functional assays. Overall, this study demonstrated how stem cell-based approaches can be used for toxicological screening and provided a valuable resource that profiles the epitranscriptomic response, which was complemented with RNA sequencing and functional and biochemical assays. This study provides relevant toxicological insights into the context of human health.

Keywords:

Papers of special note have been highlighted as: • of interest

References

1. Rowdhwal SSS, Chen J. Toxic effects of di-2-ethylhexyl phthalate: an overview. Biomed. Res. Int. 2018, 1750368 (2018).
MedlineGoogle Scholar
2. Jamarani R, Erythropel H, Nicell J, Leask R, Marić M. How green is your plasticizer? Polymers 10(8), 834 (2018).
Google Scholar
3. Hanioka N, Takahara Y, Takahara Y, Tanaka-Kagawa T, Jinno H, Narimatsu S. Hydrolysis of di-n-butyl phthalate, butylbenzyl phthalate and di(2-ethylhexyl) phthalate in human liver microsomes. Chemosphere 89(9), 1112–1117 (2012).
Medline CASGoogle Scholar
4. Serrano SE, Braun J, Trasande L, Dills R, Sathyanarayana S. Phthalates and diet: a review of the food monitoring and epidemiology data. Environ. Health 13(1), 43 (2014).
MedlineGoogle Scholar
5. Kato K, Silva MJ, Reidy JA et al. 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(3), 327–330 (2004).
Medline CASGoogle Scholar
6. Silva MJ, Barr DB, Reidy JA et al. Urinary levels of seven phthalate metabolites in the U.S. population from the National Health and Nutrition Examination Survey (NHANES) 1999–2000. Environ. Health Perspect. 112(3), 331–338 (2004).
Medline CASGoogle Scholar
7. Hauser R, Meeker JD, Singh NP et al. DNA damage in human sperm is related to urinary levels of phthalate monoester and oxidative metabolites. Hum. Reprod. 22(3), 688–695 (2007).
Medline CASGoogle Scholar
8. Pan G, Hanaoka T, Yoshimura M et al. Decreased serum free testosterone in workers exposed to high levels of di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP): a cross-sectional study in China. Environ. Health Perspect. 114(11), 1643–1648 (2006).
Medline CASGoogle Scholar
9. Muczynski V, Lecureuil C, Messiaen S et al. Cellular and molecular effect of MEHP involving LXRα in human fetal testis and ovary. PLOS ONE 7(10), e48266 (2012).
Medline CASGoogle Scholar
10. Latini G, De Felice C, Presta G et al. In utero exposure to di-(2-ethylhexyl)phthalate and duration of human pregnancy. Environ. Health Perspect. 111(14), 1783–1785 (2003).
Medline CASGoogle Scholar
11. Kambia K, Dine T, Gressier B, Dupin-Spriet T, Luyckx M, Brunet C. Evaluation of the direct toxicity of trioctyltrimellitate (TOTM), di(2-ethylhexyl) phthalate (DEHP) and their hydrolysis products on isolated rat hepatocytes. Int. J. Artif. Organs 27(11), 971–978 (2004).
Medline CASGoogle Scholar
12. Rael LT, Bar-Or R, Ambruso DR et al. Phthalate esters used as plasticizers in packed red blood cell storage bags may lead to progressive toxin exposure and the release of pro-inflammatory cytokines. Oxid. Med. Cell Longev. 2(3), 166–171 (2009).
MedlineGoogle Scholar
13. Faouzi MA, Dine T, Gressier B et al. Exposure of hemodialysis patients to di-2-ethylhexyl phthalate. Int. J. Pharm. 180(1), 113–121 (1999).
Medline CASGoogle Scholar
14. Crocker JFS, Safe SH, Acott P. Effects of chronic phthalate exposure on the kidney. J. Toxicol. Environ. Health 23(4), 433–444 (1988).
Medline CASGoogle Scholar
15. Halden RU. Plastics and health risks. Annu. Rev. Public Health 31(1), 179–194 (2010).
MedlineGoogle Scholar
16. Reilly M, Bruno CD, Prudencio TM et al. Potential consequences of the red blood cell storage lesion on cardiac electrophysiology. J. Am. Heart Assoc. 9(21), 017748 (2020).
Google Scholar
17. Jaimes R, McCullough D, Siegel B et al. Plasticizer interaction with the heart: chemicals used in plastic medical devices can interfere with cardiac electrophysiology. Circ. Arrhythm. Electrophysiol. 12(7), e007294 (2019).
Medline CASGoogle Scholar
18. Zhao J-F, Hsiao S-H, Hsu M-H et al. Di-(2-ethylhexyl) phthalate accelerates atherosclerosis in apolipoprotein E-deficient mice. Arch. Toxicol. 90(1), 181–190 (2016).
Medline CASGoogle Scholar
19. Liu N, Jiang L, Sun X et al. Mono-(2-ethylhexyl) phthalate induced ROS-dependent autophagic cell death in human vascular endothelial cells. Toxicol. In Vitro 44, 49–56 (2017).
MedlineGoogle Scholar
20. Ban J-B, Fan X-W, Huang Q et al. Mono-(2-ethylhexyl) phthalate induces injury in human umbilical vein endothelial cells. PLOS ONE 9(5), e97607 (2014).
MedlineGoogle Scholar
21. An L. Exposure to mono (2-ethylhexyl) phthalate facilitates apoptosis and pyroptosis of human endometrial microvascular endothelial cells through NLRP3 inflammasome. J. Appl. Toxicol. 2021(41), 755–764 (2020).
Google Scholar
22. Boo SH, Kim YK. The emerging role of RNA modifications in the regulation of mRNA stability. Exp. Mol. Med. 52(3), 400–408 (2020).
Medline CASGoogle Scholar
23. Wang X, ZhaoB,Roundtree I et al. N6-methyladenosine Modulates Messenger RNA Translation Efficiency. Cell 161(6), 1388–1399 (2015).
Medline CASGoogle Scholar
24. Shi H, Wei J, He C. Where, when, and how: context-dependent functions of RNA methylation writers, readers, and erasers. Mol. Cell 74(4), 640–650 (2019). • This article discusses the various aspects of epitranscriptomic regulation mediated through N6-methyladenosine modification.
Medline CASGoogle Scholar
25. Zhou KI, Parisien M, Dai Q et al. N⁶-methyladenosine modification in a long noncoding RNA hairpin predisposes its conformation to protein binding. J. Mol. Biol. 428(5, Part A), 822–833 (2016).
Medline CASGoogle Scholar
26. Engel M, Eggert C, Kaplick PM et al. The role of m⁶A/m-RNA methylation in stress response regulation. Neuron 99(2), 389–403.e389 (2018).
Medline CASGoogle Scholar
27. Jia G, Fu Y, He C. Reversible RNA adenosine methylation in biological regulation. Trends Genet. 29(2), 108–115 (2013).
Medline CASGoogle Scholar
28. Han Z, Xu Z, Yu Y et al. ALKBH5-mediated m⁶A mRNA methylation governs human embryonic stem cell cardiac commitment. Mol.Ther. Nucleic Acids 26, 22–33 (2021).
Medline CASGoogle Scholar
29. Hess ME, Hess S, Meyer KD et al. The fat mass and obesity associated gene (Fto) regulates activity of the dopaminergic midbrain circuitry. Nat. Neurosci. 16(8), 1042–1048 (2013).
Medline CASGoogle Scholar
30. Wei W, Ji X, Guo X, Ji S. Regulatory role of NV methyladenosine (m⁶A) methylation in RNA processing and human diseases. J. Cell. Biochem. 118 (9), 2534–2543 (2017).
Medline CASGoogle Scholar
31. Sayed N, Liu C, Wu JC. Translation of human-induced pluripotent stem cells. J. Am. Coll. Cardiol. 67(18), 2161–2176 (2016).
MedlineGoogle Scholar
32. Kitani T, Ong S-G, Lam Chi K et al. Human-induced pluripotent stem cell model of trastuzumab-induced cardiac dysfunction in patients with breast cancer. Circulation 139(21), 2451–2465 (2019).
Medline CASGoogle Scholar
33. Lee Won H, Ong S-G, Zhou Y et al. Modeling cardiovascular risks of e-cigarettes with human-induced pluripotent stem cell-derived endothelial cells. J. Am. Coll. Cardiol. 73(21), 2722–2737 (2019).
Medline CASGoogle Scholar
34. Le HHT, Liu C-W, Denaro P et al. Genome-wide differential expression profiling of lncRNAs and mRNAs in human induced pluripotent stem cell-derived endothelial cells exposed to e-cigarette extract. Stem Cell Res. Ther. 12(1), 593 (2021).
Medline CASGoogle Scholar
35. Doke SK, Dhawale SC. Alternatives to animal testing: a review. Saudi Pharm. J. 23(3), 223–229 (2015).
MedlineGoogle Scholar
36. Belair DG, Whisler JA, Valdez J et al. Human vascular tissue models formed from human induced pluripotent stem cell derived endothelial cells. Stem Cell Rev. Rep. 11(3), 511–525 (2015).
Medline CASGoogle Scholar
37. Ong S-B, Lee WH, Shao N-Y et al. Calpain inhibition restores autophagy and prevents mitochondrial fragmentation in a human iPSC model of diabetic endotheliopathy. Stem Cell Rep. 12(3), 597–610 (2019).
Medline CASGoogle Scholar
38. Patsch C, Challet-Meylan L, Thoma EC et al. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells. Nat. Cell Biol. 17(8), 994–1003 (2015). • This article describes the methods for endothelial cell differentiation.
Medline CASGoogle Scholar
39. Torre D, Lachmann A, Ma'ayan A. BioJupies: automated generation of interactive notebooks for RNA-seq data analysis in the cloud. Cell Syst. 7(5), 556–561 e553 (2018).
Medline CASGoogle Scholar
40. Chen EY, Tan CM, Kou Y et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics 14(1), 128 (2013).
MedlineGoogle Scholar
41. Kuleshov MV, Jones MR, Rouillard AD et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 44(W1), W90–W97 (2016).
Medline CASGoogle Scholar
42. Chen Y-H, Wu Y-J, Chen W-C et al. MEHP interferes with mitochondrial functions and homeostasis in skeletal muscle cells. Biosci. Rep. 40(4), BSR20194404 (2020).
Medline CASGoogle Scholar
43. Muczynski V, Lecureuil C, Messiaen S et al. Cellular and molecular effect of MEHP Involving LXRalpha in human fetal testis and ovary. PLOS ONE 7(10), e48266 (2012).
Medline CASGoogle Scholar
44. Tetz LM, Cheng AA, Korte CS et al. Mono-2-ethylhexyl phthalate induces oxidative stress responses in human placental cells in vitro. Toxicol. Appl. Pharmacol. 268(1), 47–54 (2013).
Medline CASGoogle Scholar
45. Kuo CH, Hsieh CC, Kuo HF et al. Phthalates suppress type I interferon in human plasmacytoid dendritic cells via epigenetic regulation. Allergy 68(7), 870–879 (2013).
Medline CASGoogle Scholar
46. Meyer KD, Patil DP, Zhou J et al. 5′ UTR m(6)A promotes cap-independent translation. Cell 163(4), 999–1010 (2015).
Medline CASGoogle Scholar
47. Chen M, Wong C-M. The emerging roles of N⁶-methyladenosine (m⁶A) deregulation in liver carcinogenesis. Mol. Cancer 19(1), 44 (2020).
Medline CASGoogle Scholar
48. Li T, Hu P-S, Zuo Z et al. METTL3 facilitates tumor progression via an m⁶A-IGF2BP2-dependent mechanism in colorectal carcinoma. Mol. Cancer 18(1), 112 (2019).
MedlineGoogle Scholar
49. Jiang H, Cao K, Fan C, Cui X, Ma Y, Liu J. Transcriptome-wide high-throughput m⁶A sequencing of differential m⁶A methylation patterns in the human rheumatoid arthritis fibroblast-like synoviocytes cell line MH7A. J. Inflamm. Res. 14, 575–586 (2021).
MedlineGoogle Scholar
50. Meeker JD, Sathyanarayana S, Swan SH. Phthalates and other additives in plastics: human exposure and associated health outcomes. Philos. Trans. R. Soc. Lond. B Biol. Sci. 364(1526), 2097–2113 (2009).
Medline CASGoogle Scholar
51. Huang S, Qi Z, Ma S, Li G, Long C, Yu Y. A critical review on human internal exposure of phthalate metabolites and the associated health risks. Environ. Pollution 279, 116941 (2021).
Medline CASGoogle Scholar
52. Hauser R, Calafat AM. Phthalates and human health. Occup. Environ. Med. 62(11), 806 (2005).
Medline CASGoogle Scholar
53. Lu X, Xu X, Lin Y, Zhang Y, Huo X. Phthalate exposure as a risk factor for hypertension. Environ. Sci. Pollution Res. 25(21), 20550–20561 (2018).
Medline CASGoogle Scholar
54. Mariana M, Feiteiro J, Verde I, Cairrao E. The effects of phthalates in the cardiovascular and reproductive systems: a review. Environ. Int. 94, 758–776 (2016).
Medline CASGoogle Scholar
55. Campioli E, Lau M, Papadopoulos V. Effect of subacute and prenatal DINCH plasticizer exposure on rat dams and male offspring hepatic function: the role of PPAR-α. Environ. Res. 179, 108773 (2019).
Medline CASGoogle Scholar
56. Caldwell JC. DEHP: genotoxicity and potential carcinogenic mechanisms – a review. Mutat. Res. Rev. Mutat. Res. 751(2), 82–157 (2012).
CASGoogle Scholar
57. Jiang X, Liu B, Nie Z et al. The role of m⁶A modification in the biological functions and diseases. Signal Transduct. Target. Ther. 6(1), 1–16 (2021).
MedlineGoogle Scholar
58. Liu X, Wang H, Zhao X et al. Arginine methylation of METTL14 promotes RNA N⁶-methyladenosine modification and endoderm differentiation of mouse embryonic stem cells. Nat. Commun. 12(1), 3780 (2021).
Medline CASGoogle Scholar
59. Yu F, Wei J, Cui X et al. Post-translational modification of RNA m⁶A demethylase ALKBH5 regulates ROS-induced DNA damage response. Nucleic Acids Res. 49(10), 5779–5797 (2021).
Medline CASGoogle Scholar
60. Du Y, Hou G, Zhang H et al. SUMOylation of the m⁶A-RNA methyltransferase METTL3 modulates its function. Nucleic Acids Res. 46(10), 5195–5208 (2018).
Medline CASGoogle Scholar
61. Tesauro M, Mauriello A, Rovella V et al. Arterial ageing: from endothelial dysfunction to vascular calcification. J. Intern. Med. 281(5), 471–482 (2017).
Medline CASGoogle Scholar
62. Kataria A, Levine D, Wertenteil S et al. Exposure to bisphenols and phthalates and association with oxidant stress, insulin resistance, and endothelial dysfunction in children. Pediatr. Res. 81(6), 857–864 (2017).
Medline CASGoogle Scholar
63. Bai C, Liu L, Chen S et al. Urinary phthalate metabolites and arterial stiffness: a panel study. Environ. Res. 207, 112657 (2022).
Medline CASGoogle Scholar
64. Nardelli TC, Erythropel HC, Robaire B. Toxicogenomic screening of replacements for di(2-ethylhexyl) phthalate (DEHP) using the immortalized TM4 sertoli cell line. PLOS ONE 10(10), e0138421 (2015).
MedlineGoogle Scholar
65. Roy D, Morgan M, Yoo C et al. Integrated bioinformatics, environmental epidemiologic and genomic approaches to identify environmental and molecular links between endometriosis and breast cancer. Int. J. Mol. Sci. 16(10), 25285–25322 (2015).
Medline CASGoogle Scholar
66. Aryal B, Singh AK, Zhang X et al. Absence of ANGPTL4 in adipose tissue improves glucose tolerance and attenuates atherogenesis. JCI Insight 3(6), e97918 (2018).
MedlineGoogle Scholar
67. Aryal B, Price NL, Suarez Y, Fernández-Hernando C. ANGPTL4 in metabolic and cardiovascular disease. Trends Mol. Med. 25(8), 723–734 (2019).
Medline CASGoogle Scholar
68. Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149(7), 1635–1646 (2012). • This article discusses the landscape of N6-methyladenosine epitranscriptomic modification.
Medline CASGoogle Scholar
69. Berulava T, Buchholz E, Elerdashvili V et al. Changes in m⁶A RNA methylation contribute to heart failure progression by modulating translation. Eur. J. Heart Fail. 22(1), 54–66 (2020).
Medline CASGoogle Scholar

Vol. 14, No. 19

Supplemental Materials

Metrics

Downloaded 138 times

History

Received 3 April 2022

Accepted 30 August 2022

Published online 31 October 2022

Published in print October 2022

Information

Keywords

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.futuremedicine.com/doi/suppl/10.2217/epi-2022-0110

Author contributions

J Jousma carried out bioinformatic analysis, data visualization, cellular and molecular experimental methods as well as manuscript preparation and revisions. SB Nukala contributed to cellular experimental methods. Z Han contributed to cellular experimental methods. G Yan and HHT Le carried out validation of statistical analysis. Y Kwon and Y Li contributed cellular experimental methods. WH Lee and SG Ong carried out project conceptualization, design, supervision, resources and manuscript review. All authors read and approved the final manuscript. SB Ong made intellectual contributions during review and preparation.

Financial & competing interests disclosure

This paper was supported and funded by the National Heart, Lung, and Blood Institute (NHLBI) T32 HL007829 (JJ), (NHLBI) R00 HL130416 (S-GO) and R01 HL148756 (S-GO). The authors also received funding from the American Heart Association (AHA), Postdoctoral Fellowship 917176 (ZH), AHA Scientist Development Grant 16SDG27560003 (WHL). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Data availability

RNA sequencing and N6-methyladenosine microarray data will be made publicly accessible upon request to the corresponding authors and will be available in the Gene Expression Omnibus database at the accession number GSE214420.

PDF download

Alteration of the N6-methyladenosine epitranscriptomic profile in synthetic phthalate-treated human induced pluripotent stem cell-derived endothelial cells

Abstract

Plain language summary

References

Alteration of the N⁶-methyladenosine epitranscriptomic profile in synthetic phthalate-treated human induced pluripotent stem cell-derived endothelial cells