Effects of phthalate exposure on asthma may be mediated through alterations in DNA methylation

I-Jen Wang, Wilfried JJ Karmaus, Su-Lien Chen, John W Holloway, Susan Ewart

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. 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. Effects of phthalate exposure on asthma may be mediated through alterations in DNA methylation.