title: Effects of phthalate exposure on asthma may be mediated through alterations in DNA methylation.
abstract: BACKGROUND
  Phthalates may increase the asthma risk in children. Mechanisms underlying this association remain to be addressed. This study assesses the effect of phthalate exposures on epigenetic changes and the role of epigenetic changes for asthma. In the first step, urine and blood samples from 256 children of the Childhood Environment and Allergic diseases Study (CEAS) were analyzed. Urine 5OH-MEHP levels were quantified as an indicator of exposure, and asthma information was collected. DNA methylation (DNA-M) was measured by quantitative PCR. In the screening part of step 1, DNA-M of 21 potential human candidate genes suggested by a toxicogenomic data were investigated in 22 blood samples. Then, in the testing part of step 1, positively screened genes were tested in a larger sample of 256 children and then validated by protein measurements. In step 2, we replicated the association between phthalate exposure and gene-specific DNA-M in 54 children in the phthalate contaminated food event. In step 3, the risk of DNA-M for asthma was tested in 256 children from CEAS and corroborated in 270 children from the Isle of Wight (IOW) birth cohort.
  
  
  RESULTS
  Differential methylation in three genes (AR, TNFα, and IL-4) was identified through screening. Testing in 256 children showed that methylation of the TNFα gene promoter was lower when children had higher urine 5OH-MEHP values (β = -0.138, P = 0.040). Functional validation revealed that TNFα methylation was inversely correlated with TNFα protein levels (β = -0.18, P = 0.041). In an additional sample of 54 children, we corroborated that methylation of the TNFα gene promoter was lower when urine 5OH-MEHP concentrations were higher. Finally, we found that a lower methylation of 5'CGI region of TNFα was associated with asthma in 256 CEAS children (OR = 2.15, 95% CI = 1.01 to 4.62). We replicated this in 270 children from the IOW birth cohort study. Methylation of the CpG site cg10717214 was negatively associated with asthma, when children had 'AA' or 'AG' genotype of the TNFα single nucleotide rs1800610.
  
  
  CONCLUSIONS
  Effects of phthalate exposure on asthma may be mediated through alterations in DNA methylation.
authors.affiliation: Department of Pediatrics, Taipei Hospital, Ministry of Health and Welfare, #127, Su-Yuan Road, Hsin-Chuang Dist 242 New Taipei City, Taiwan ; Institute of Environmental and Occupational Health Sciences, College of Medicine, National Yang-Ming University, Taipei, 112 Taiwan ; Department of Health Risk Management, China Medical University, Taichung, 404 Taiwan.
authors.forename: I-Jen
authors.initials: IJ
authors.lastname: Wang
authors.affiliation: Division of Epidemiology, Biostatistics, and Environmental Health, School of Public Health, University of Memphis, Memphis, 38152 USA.
authors.forename: Wilfried Jj
authors.initials: WJ
authors.lastname: Karmaus
authors.affiliation: Genomics, BioSci & Tech, Taipei, 221 Taiwan.
authors.forename: Su-Lien
authors.initials: SL
authors.lastname: Chen
authors.affiliation: Clinical and Experimental Sciences, Faculty of Medicine, and NIHR Respiratory Biomedical Research Unit, University of Southampton, Southampton, S016 6YD UK ; Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, SO16 6YD UK.
authors.forename: John W
authors.initials: JW
authors.lastname: Holloway
authors.affiliation: Department of Large Animal Clinical Sciences, Michigan State University, East Lansing, 48824 MI USA.
authors.forename: Susan
authors.initials: S
authors.lastname: Ewart
keywords: Asthma; DEHP exposure; DNA methylation; Quantitative PCR; TNFα