| Summary: | 碩士 === 國立成功大學 === 環境醫學研究所 === 99 === Rising prevalence and morbidity of childhood asthma and allergic diseases has been evident throughout the world in recent years. Studies have shown that, both the genetics and biological pollutants, such as dust mites, and fungal spores in indoor and outdoor environment, are major risk factors for the development and exacerbation of allergic diseases. However, as a multi-factorial disease, the rising prevalence mentioned can’t be completely justified only by those factors. Consequently, the increasing presence of emerging chemical pollutants in overall environment since the industrial era is considered one important risk factor in modern society. Phthalate esters (PAEs), a kind of chemical materials used wildly in the PVC products have been reported in previous epidemiology studies that phthalates exposure might have adverse effects on respiratory symptoms and cause allergic diseases in children. However, the mechanism between phthalates exposure and allergy/asthma were not confirmed clearly in the animal studies, and there were not sure that phthalates exposure could trigger an adjuvant effect of TH2 immune response or induce allergic reaction by inflammation mechanism. This study is therefore aimed to investigate the underlying mechanism by assessing the associations between phthalates exposure, using urinary metabolites levels, and also consider other environmental factors to try to understand clearly about the effect of allergic-related and inflammatory-related cytokines in children.
Study subjects of pre-school children were selected from our previous questionnaires survey. According the standardized inclusion and exclusion criteria to select subjects and then divided into 980 cases and 801controls. All the selected subjects were asked by telephone for participating in the follow-up study, and then about 27% cases and 11% control subjects agreed. To further invited participators to the medical center of the great metropolitan for clinic visit to confirm their health status and collected the serum samples to analyze the levels of biomarkers. Moreover, we investigated the environmental exposure by standardized questionnaires, house dust and other biological and chemical sample collection to understand the levels about children exposure, and the children’s urine samples also collected during households’ visits. We measured the levels of children’s phthalates exposure about DMP (metabolite: MMP), DEP (metabolite: MEP), DBP (metabolite: MBP), BBzP (metabolite: MBzP), DEHP (metabolite: MEHP, MEHHP and MEOHP) in children’s urine by HPLC-MS/MS, and the levels of eight cytokines including TH1 pathway-related cytokines (IL-12 and IFN-γ), the TH2 pathway-related cytokines (IL-4, IL-5 and IL-13) and inflammatory-related cytokines (IL-6, IL-8 and TNF-α) in serum were also measured by flow cytometry. Finally, total of 101 children had completely information about healthy states and exposure profiles, and the data would analyze by statistics methods. We first divided the children into high or low exposure group based on the median of total PAEs’ metabolites levels in children’s urine estimated by the summary of individual PAE. Relationship between each PAE and the levels of cytokines was further also assessed by defining the subject group with high phthalates exposure was those having the phthalates’ metabolites levels above the median of all phthalates’ metabolites levels measured in urine (MMP: 4.87, MEP: 12.83, MBP: 57.93, MBzP: 4.78, MEHP: 8.66, MEHHP: 42.34, MEOHP: 40.55 ng/ml). On the contrary, the levels of phthalates’ metabolites below the median of phthalates’ metabolites levels in urine were defined the low exposure groups.
In general, children with higher levels of total PAEs in urine were found to have higher level of IL-5 (Hightotal: 1.30 vs. Lowtotal: 0.55 pg/ml, p=0.04) and TNF-α (Hightotal: 1.12 vs. Lowtotal: 0.90 pg/ml, p=0.03) in serum. For each PAE, children with higher levels of MBP and MEHP in urine, who had higher level of IFN-γ in serum (HighMBP: 1.15 vs. LowMBP: 0.35 pg/ml, p=0.03; HighMEHP: 1.13 vs. LowMEHP: 0.35 pg/ml, p=0.04), and higher levels of IL-5 (HighMEHHP: 1.30 vs. LowMEHHP: 0.55 pg/ml, p=0.02; HighMEOHP: 11.30 vs. LowMEOHP: 0.55 pg/ml, p=0.02), IL-8 (HighMEHHP: 2.15 vs. LowMEHHP: 1.96 pg/ml, p=0.02; HighMEOHP: 2.15 vs. LowMEOHP: 1.96 pg/ml, p=0.05) and TNF-α (HighMEHHP: 1.25 vs. LowMEHHP: 0.90 pg/ml, p=0.01; HighMEOHP: 1.12 vs. LowMEOHP: 0.90 pg/ml, p=0.01) in serum were also showed in higher levels of MEHHP and MEOHP’s exposure group children. To further estimate the cytokines’ level in serum between allergic and healthy children in high and low exposure groups, respectively. In higher group, allergic children with higher levels of MBP in urine, who had higher level of IL-5 (CaseMBP: 1.36 vs. ControlMBP: 0.55 pg/ml, p=0.01) and TNF-α (CaseMBP: 1.40 vs. ControlMBP: 0.35 pg/ml, p=0.001) when compared to healthy children. On the contrary, allergic children compared with healthy in low exposure group, data were showed that the level of IL-5 (CaseMEHP: 1.23 vs. ControlMEHP: 0.55 pg/ml, p=0.04) were higher in allergic children in MEHP exposure, and then levels of TNF-α on 75 percentile (CaseMEHHP: 1.47 vs. ControlMEHHP: 0.35 pg/ml, p=0.01; CaseMEOHP: 1.51 vs. ControlMEOHP: 0.35 pg/ml, p=0.03) also present the allergic children had higher level than health children in MEHHP and MEOHP. Moreover, we also found that phthalate exposures were associated with the levels of IL-8 and TNF-α no matter in allergic children or healthy children’s serum. Therefore, our results were showed that phthalates exposure may cause allergy-related and inflammatory reactions happened. According to the Spearman's rank correlation coefficient to estimate the relationship between phthalate exposure and children’s cytokines profiles in serum, data were showed that levels of MEHHP and MEOHP in urine were associated with TH2 pathway-related cytokines IL-5 (r=0.30, p =0.03; r=0.30, p=0.03) and inflammation-related cytokine TNF-α (r=0.31, p=0.002 ; r=0.30, p=0.002). We also used the logistic regression model to evaluate the odds ratio for phthalates exposure and cytokines profiles (IL-5, IL-8, IFN-γ and TNF-α), and then adjusted by other environmental risk factors. Results were showed that the DEHP’s metabolite MEHHP still play a risk factor to IL-5 (aOR=2.84, 95%CI: 1.05-7.70), IFN-γ (aOR=4.18, 95%CI: 1.18-14.79) and IL-8 (aOR=2.83, 95%CI: 1.01-7.91) in serum.
In conclusions, our study showed that phthalates exposure, especially DBP and DEHP exposure, would induce the inflammatory-related cytokines, such as TNF-α secretion. On the other hand, allergic children would also have higher inflammatory-related and allergic-related cytokines, while they exposed to phthalates. We therefore propose, with quantitative evidence from field study, that phthalates exposure may be more likely to be linked to inflammatory of immune response and made mixed TH1/TH2 adjuvant effect in allergic children, implying that the levels of PAEs were related to the level of IL-5 and IFN-γ in serum.
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