The use of harmful chemicals has been increasing in modern society. There has also been a simultaneous increase in the number of asthma and other allergic disorders in children. Global Initiative of Asthma reported in 2004 that as many as 300 million people of all ages have asthma, and the estimated number might be further increased in the future [1
In Taiwan, the prevalence of children affected by asthma was around 12%; asthma is one of the most important health issues in children in Taiwan and worldwide [2
]. Our previous studies and those of others have shown that children with asthma have difficulty in learning due to hyperactive and impulsive behaviors that are secondary consequences of the illness [4
], resulting in a considerable burden on public health.
BPA is an endocrine-disrupting chemical being increasingly used in modern society. Hence, health concerns regarding BPA use should not be ignored [6
]. Some epidemiological studies have presented the association between BPA exposure and a higher prevalence of asthma [7
]. Pulmonary pathological data further support that BPA might aggravate asthma through adjuvant effects [8
]. In our previous study, we discovered that BPA exposure was related to high IgE levels in children [9
]. We also discovered that the urine BPA metabolite level was significantly higher in Taiwanese children than in those of other countries [9
]. BPA has become serious public health problem globally and been described the various epigenetic mechanisms, like DNA methylation, histone modifications and non-coding RNAs, then affecting gene expression [11
]. Furthermore, we found that exposure to this harmful chemical affected DNA methylation, which influenced the prevalence of allergic disorders in children [14
]. Therefore, epigenetic variations caused by environmental factors might be associated with the development of asthma.
Multiple exposure routes including oral, dermal, and airway routes contribute to the total intracellular BPA concentration in children [15
]. The increasing health concern was attributed to the continuous low-level exposure to BPA in children [15
]. BPA is not bio-accumulative, but continuous exposure from many sources makes it as harmful as a bio-accumulative compound [18
]. According to a recent report, BPA has a longer than expected half-life [16
]. Some animal studies have reported that several endocrine-disrupting environmental chemicals can modify epigenetic marks [19
]. Several studies have focused on the epigenetic modifications caused by environmental exposure to BPA, but most of them have not been directly linked to a clinical endpoint [20
]. Further, there is limited information available from large-scale data regarding children exposed to BPA based on clinical specimens. In this study, we investigated the effects of BPA exposure on epigenetic modification and established a correlation between healthy and asthmatic children. This study focused on whether exposure to BPA induces aberrant DNA methylation of specific genes related to childhood asthma in children aged 3 years.
This study is the first large-scale assessment demonstrating a link between BPA exposure and asthma via epigenetic mechanisms in children. We demonstrated that a higher exposure to BPA is related to a lower DNA methylation of the MAPK1 gene, which is associated with a higher risk of developing asthma. Furthermore, we showed that the protein level of MAPK1 were inversely related to BPA exposure.
It has been shown that BPA is a harmful environmental chemical and its exposure may influence the human immune system [8
]. BPA exposure might affect the immune system by releasing some pro-inflammatory mediators, including cysteinyl leukotriene, MAPK1, prostaglandin D2, and IL-13, which might be related to the development of asthma [32
]. In addition, BPA exposure has been shown to affect many human chronic diseases, including diabetes, metabolic syndrome, reproductive disorders, cardiovascular diseases, respiratory diseases, and breast cancer [33
]. Animal studies suggested that BPA exposure might reduce the levels of regulatory T cells, IL-10, and IFN-γ and increase the production of IL-4 and antigen-specific IgE [34
]. Donohue et al. (2012) reported that urinary BPA levels at the ages of 3, 5, and 7 years were associated with childhood asthma between the ages of 5 and 12 years [7
]. Pre-natal and post-natal BPA exposure has been reported to increase the odds of childhood asthma and allergic disorders [7
]. Moreover, our previous study showed that higher BPA exposure was associated with increased serum IgE levels and might be related to the development of allergic disorders, particularly in children [9
]. However, the mechanism underlying BPA-induced asthma remains unknown.
In our study, we found that BPA exposure was related to decreased methylation of MAPK1
5′CGI, which might be related to the development of asthma in children. Microtubule Affinity Regulating Kinase 1 (MARK1
) is a protein-coding gene and a member of the MAPK signaling pathway. Among its functions are cytoskeletal signaling, energy metabolism, production of pro-inflammatory mediators, and cell growth and differentiation. Hung et al. demonstrated that circulating myeloid dendritic cells treated with two common environmental endocrine-disrupting chemicals, nonylphenol, and 4-octylphenol, increased the expression of tumor necrosis factor-α via the MAPK signaling pathway [41
]. Another study showed that overexpression of a family of MAPKs in cells leads to hyperphosphorylation of microtubule-associated proteins and disruption of the microtubule array, resulting in morphological changes and cell death [42
]. These findings indicate that exposure to endocrine disruptors can potentially alter the DNA methylation status of the MARK1
Furthermore, prenatal exposure to BPA was reported to alter the methylation status of the genes related to reproductive processes in the animal [43
] and human [38
]. In addition, BPA could induce a dose-dependent activation of the pro-inflammatory cytokine MAPK1 and the formation of reactive oxygen species (ROS) in rat alveolar macrophages [42
]. Therefore, BPA exposure might trigger airway macrophages to express MAPK1
, mediated by alterations in DNA methylation, which then activates downstream signals to enhance inflammatory responses [47
Higher BPA exposure was shown to lead to a decrease in MAPK1
5′CGI methylation in our study. Lower methylation of MAPK1
5′CGI is associated with increased gene expression, which in turn increases MAPK1
expression at protein levels and triggers the development of allergic inflammation [49
]. We demonstrated that the methylation of MAPK1
5′CGI is an intervening factor between BPA exposure and asthma. MAPK signaling pathways are known to be involved in airway inflammation and the regulation of immune cells, which are the hallmark features of asthma [49
]. In addition, genetic variants of MAPK1
might be involved in regulating cytokine levels in asthma patients, which might modulate the severity of asthma [54
We initially found DNA methylation in the AR gene, which is related to BPA exposure. However, AR failed to display a significant difference in methylation after adjusting for potential confounders. In particular, sex differences in AR expression may also account for this finding.
There are a few limitations to our study. First, our study was confined to the use of the candidate genes approach. The candidate genes approach has limited accuracy due to the dependence on prior studies, which lead to an information bottleneck. Therefore, instead of only one candidate gene, we chose 33 candidate genes from a published toxicogenomics database to carry out our investigations. This approach provides biological plausibility to our study and is more cost-effective than the genome-wide approach. Second, RNA samples were not available for this study. High-quality RNA isolation from blood was technically difficult because of its limited cellular components. However, we assessed the relationship between the methylation and translation of genes via protein quantitation. Third, our analysis was based on a single morning urine sample. However, spot urine samples and 24-h urine samples have been reported to produce similar levels of daily BPA intake [57
]. Even though BPA has a relatively short half-life, its continuous daily intake contributes to an exposure scenario that is similar to those of bioaccumulative compounds [58
]. Additionally, if the measurements are not precisely and carefully performed, outcomes are nullified and the effects of exposure could be underestimated. Forth, the DNA-methylation information is derived from whole blood samples, which includes other cell types besides lymphocytes. The whole blood consists of many functionally distinct cell populations. The interpretation of DNA-methylation profiles from whole blood should be conducted with great caution, because the differences might be resulted from varying proportions of white blood cell types. In this study, we analyzed DNA-methylation status of 33 genes in whole blood DNA, and found methylation status of MAPK1
gene was associated with BPA exposure. However, the FACS sorting approach might be needed to isolate neutrophils, B and T lymphocytes, monocytes, and other granulocytes to study which subpopulation of whole blood cells are affected.
The strengths of this study are the collection of clinical specimens with available clinical and environmental exposure data. The use of urinary BPAG level analyzed by UPLC-MS/MS provided a more direct measure of individual BPA exposure. Furthermore, asthma was confirmed by pediatric allergists using a questionnaire. Diagnosis of asthma by pediatric allergists through questionnaires is a gold standard method [60
]. Therefore, the errors in the classification of the outcome could be minimized through these means. Another strength of our study is that we employed a two-step study: MDFS method was used to identify candidate gene methylation and was then followed by qPCR to confirm the methylation status of the candidate genes in a larger sample cohort of 228 children. This step-wise approach allowed us to identify the possible candidates in a cost-effective manner.