Association among phthalate exposure, DNA methylation of RUNX3, and childhood allergy

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(1)Association among phthalate exposure, DNA methylation of RUNX3, and childhood allergy Wan-Ru. a Wang ,. Nai-Yun. a Hsu ,. I-Ying. b Kuo ,. Huey-Jen. a Su. aDepartment. of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan, Taiwan bDepartment of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.  Introduction. Research has shown that exposure to phthalate esters, one widely used plasticizer, are related to the development of allergic morbidities. Environmental exposures could influence health status via the epigenetic regulations. Runt-related transcription factor 3 (RUNX3) is found to bind with FOXP3 promoter, and then increase the level of regulator T cell. On the other hand, thymic stromal lymphopoietin (TSLP) genetic dysregulated expression have been linked to the presence of atopic diseases such as atopic dermatitis, asthma, allergic rhinitis and eosinophilic esophagitis as well as some immune-mediated diseases. This study is aimed to verify the effects of phthalate exposures on the changes of gene methylation on RUNX3 and TSLP..  Method and material. A total of 90 children recruited from the Greater Tainan area during 2005 to 2006 were selected in current analysis. Each of them had detailed health examination for allergic status, and was investigated for the exposure level of di-2-ethylhexyl phthalate (DEHP) in settled dusts of their bedroom. Metabolites of DEHP including mono-2-ethylhexyl phthalate (MEHP), mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono-(2ethyl-5-oxohexyl) phthalate (MEOHP) in urine (µg/g creatinine) were also measured. Their serum samples were used to examine the total IgE level and DNA methylation status. Methylation on the gene promoters of RUNX3 (one promoter site) and TSLP (four promoter sites) was analyzed by the SYBR Green-based quantitative methylationspecific PCR (qMSP) and pyrosequencing (PyroMark Q24, Qiagen), respectively. The percentage of methylation level (Met%) derived from qMSP method was based on the equation: [1 / (1 + 2 − (Ctum − Ctm)) × 100%, where Ctum is the threshold of average cycle (Ct) obtained from duplicate qPCRs using the pair of un-methylated primers, and Ctm is the average Ct acquired using the pair of methylated primers. Once the Ct values of both of the primer pairs for un-methylated and methylation were over 35, the data would not be considered in the analysis due to the levels were too low. For those Met% level less than 1% were considered as 1% in data analysis.. Figure 1. The flow chart of this study.  Results and discussion. Study subjects were about 7.04 ± 1.19 years old and nearly 56% were female. Almost 79% of their parents did not smoke. The medium level of met% for four TSLP and one RUNX3 were 17.50%, 19.00%, 38.00%, 17.00%, and 12.88%, respectively (Table 1). Lower TSLP met% level on the promotor-838 was associated with the higher quartile of DEHP exposure level (Figure 1). Negative association of levels between TSLP met% and dust borne DEHP was also shown (Table 2). It is suggested that phthalate exposure would increase the TSLP expression, and might link to the presence of allergic diseases. On the other hand, inverse relationship between the levels of RUNX3 met% and total metabolites of DEHP was observed (Table 2). The lower RUNX3 met% represents the higher expression of RUNX3 gene, and the level of regulator T cell would therefore increase. The increasing regulator T cell is associated with rising level of CD4+ T cell which might result in the development of allergic disease. Table 1. DEHP concentration and met%. Category Parent compounds in settled dust (µg/g dust) Sum of metabolites in urine (μg/g Cr) MEHP MEHHP MEOHP TSLP Met(%) promotor -838 promotor -842 promotor -860 promotor -863 RUNX3 Met (%) promotor -308 to -288. N 81 90 90 90 90. Medium (25th-75th) 1219.63 (1053.992143.73) 100.77 (102.08-306.95) 10.11 (10.87-27.89) 45.12 (46.65-152.17) 42.62 (44.2-127.24). 90 90 90 90. 17.50 (22.26-30.03) 19 (24.07-31.19) 38 (39.77-46.25) 17 (30.93-45.67). 73. 12.88 (23.17-39.52). Table 2. Correlations of levels between DEHP exposures and gene methylations Parental compounds in Correlation settled dusts, μg/g dust Methylation level of TSLP CpG (%) promotor -838 -0.241* promotor -842 -0.159 promotor -860 -0.206 promotor -863 -0.244* Methylation level of RUNX3 CpG (%) promotor -308 to -288 0.186 coefficient¶. ¶ spearman????;. TSLP promotor -838 vs. DEHP p-value = 0.014*. DEHP in settle dust (µg/g dust) (c). TSLP promotor -860 vs. DEHP p-value = 0.153. -0.246*. *Sum of levels of MEHP, MEHHP and MEOHP. DEHP in settle dust (µg/g dust). (d) TSLP promotor -863 vs. DEHP p-value = 0.194. DEHP in settle dust (µg/g dust). Metabolism in urines, μg/g Cr 0.011 0.09 0.126 0.44. TSLP promotor -842 vs. DEHP p-value = 0.194. (b). (a).  Conclusion. DEHP in settle dust (µg/g dust). (e) RUN3 vs. DEHP p-value = 0.223. *. Fig 2. DEHP in settle dust, metabolite of DEHP and DNA methylation. (a) Boxplot of quartile of DEHP and methylation of TSLP promotor -838, (b) Boxplot of quartile of DEHP and methylation of TSLP promotor -842, (c) Boxplot of quartile of DEHP and methylation of TSLP promotor -860, (d) Boxplot of quartile of DEHP and methylation of TSLP promotor -863, (e) Boxplot of quartile of sum of DEHP metabolite and methylation of RUNX3 ¶* 1st quartile=0-25%, 2nd quartile=25-50%, 3rd quartile=50-75%, 4th quartile=75-100%, DEHP in settle dust: 25 percentile=776.12, 50 percentile=1260.49, 75 percentile=1845.73 RUNX3: 25 percentile=51.9, 50 percentile=100.77, 75 percentile=180.02. It is found that the exposure to DEHP collected in dusts might result in the decrease of TSLP methylation. In addition,¶ the higher metabolite concentration was related to the lower RUNX3 methylation level. The results of this study could contribute to the understanding of the etiology of phthalate-related biomarker and may initiate new strategies for an early prevention or a therapeutic intervention in children’s exposed to phthalates..

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