In this exploratory study, we have identified ACSL3 as a candidate biomarker/gene whose 5′-CGI methylation status appears to be related to transplacental PAH exposure and further associated with PAH-associated asthma. Thus, ACSL3 may be the first potential surrogate endpoint for environmentally related childhood asthma. If confirmed, such a biomarker holds promise in assessing PAH exposure and as a clinically relevant predictor for asthma risk in children born to mothers exposed to air pollutants such as traffic-related combustion emissions.
We used a set of stringent criteria for the identification, selection and validation of candidates, which resulted in choosing ACSL3 the final candidate for the 56-sample study. We first used an unbiased method, MSRF, to identify both hypo- and hyper-methylated sequences in the high PAH group compared to the low PAH group. In silico analyses then revealed that 19 out of 31 sequences identified were homologous to known genes and 6 of these aligned to a CGI(s) located in the 5′ flanking region of a known gene (5′-CGI). These 6 genes therefore were chosen as putative candidates for further investigation. By comparing the degree of methylation of their 5′CGI in UCWBC DNA samples and levels of gene expression in FPT RNA samples we identified ACSL3 to have the highest inverse concordance in vivo. We then employed a lung cancer cell line (H1299) to demonstrate that the demethylation of ACSL3 5′-CGI by 5-aza-dC treatment induced enhancement of gene expression in H1299. Conversely, the treatment of H1299 with low doses of BaP, a PAH, was associated with dose-dependent hypermethylaiton of ACSL3 5-CGI and gene silencing. Collectively, these data strongly support methylation of the 5′-CGI of ACSL3 in gene regulation. They also provide evidence that PAH could directly induce ACSL3 promoter methylation. This well though out, step-wise approach allowed us to identify a candidate to be tested in the 56-sample study to determine whether methylation of the ACSL3 5′CGI could be a stand alone biomarker for PAH exposure and/or PAH-associated childhood asthma.
Measuring human exposure to chemicals such as PAHs and determining their relationship to disease outcomes is a challenge for environmental health, especially when children are the study subjects. Although some studies have found a positive correlation between PAH-induced DNA adducts and mutagenesis or cancer development 
others have failed to establish clear cause-effect relations between PAH adducts and the induction of these or other disease outcomes 
, suggesting that other mechanisms may be involved. Moreover, some of the methods for measuring genotoxic mechanisms such as adducts are complex and low-throughput. Therefore, it is of significance that, in this investigative study, we have developed a relatively simple, high throughput assay based on methylation of a sequence in the promoter of the ACSL3
gene that appears to distinguish children born to mothers with high airborne PAH exposure from those who were not (OR
<0.001) with high sensitivity (75%) and specificity (85%). The Area Under ROC curve (AUC) value for this assay was 0.82 suggesting excellent separability of the subjects with the methylated or unmethylated ACSL3
sequence. This optimized MSPCR protocol allows for a rapid determination of ACSL3
5′-CGI methylation status in large sample sets, which is a normal requirement in population studies. It also has the added advantage to be developed into a minimally invasive test since only a small amount of DNA from UCWBC is required for the assay. If validated, such an assay is expected to have a high utility in the assessment of PAH exposure in future population-based studies.
Multiple epidemiological studies have suggested that asthma risk is determined by prenatal and/or early life exposure to pollutants and allergens that modify later life lung function and T helper cell allergic phenotype 
. Epigenetic modifications of regulatory genes crucial to the development of asthma-related pathophysiology have been put forth as a possible mechanistic basis of this disease 
. Thus, asthma may be caused by epigenetic changes such as DNA methylation, post-translational modifications of histones, dysregulation of non-coding RNAs, and chromatin alterations. At present, the best studied mechanism is DNA methylation-mediated disruption of gene regulation. In this regard, experimental data indicating that DNA methylation of genes critical to the differentiation of T helper cells could skew their response towards or away from an allergic phenotype lend support to this theory 
. Additionally, we recently demonstrated that inhaled diesel exposure (a source of PAHs) and intranasal Aspergillus fumigatus
induced specific DNA methylation changes in the promoters of interferon-γ
in T helper cells, respectively, and altered circulating levels of IgE in mice 
. Despite progress made in experimental studies, fundamental questions related to asthma risk among human populations remain; these can only be addressed by cohort studies using specimens linked to well-annotated clinical and environmental exposure data. Such cohort-driven epigenetic research has the potential to address key questions, such as those concerning the influence of early life factors, environmental exposure, other disease states, and lifestyle factors on susceptibility to asthma. In this proof-of-principle study, we developed an innovative approach to population-based studies of environmental epigenetics, which could easily be implemented in future large-scale investigations.
A range of approaches is available to obtain quantitative and qualitative information on genomic DNA methylation changes 
. We chose methylation sensitive restriction fingerprinting ( MSRF; 
) as a tool for the discovery of PAH-reprogrammable genes in UCWBC due to its high sensitivity, requirement for very small amounts of starting materials (~100 ng), and the ease in visual identification of differentially methylated sequences among a moderately large number of samples. Using only four arbitrary primers and six permutations of MSRF analyses on UCWBC samples from the high and low PAH exposure groups, we discovered 31 differentially methylated sequences. In future studies more arbitrary primers could be used to cover a larger portion of the genome in order to uncover more candidates 
. In this study we had overcome another technical hurdle related to the quantification of gene expression in UCWBC. This task is challenging because the amount of cellular components in umbilical cord blood is usually very limited and prohibitive for high quality RNA isolation. To circumvent this problem, in a subset of samples, we assessed the concordance between the extents of methylation in the promoter-CGI(s) of each gene (using UCWBC DNA samples) with its respective transcript expression levels in RNA samples isolated from matched FPT samples. This choice was based on the logic that both UCWBC and FPT are fetal tissues are subjected to similar environmental exposure through the mother. Our results demonstrated good concordance for at least the ACSL3
gene. In future design of such an epigenetic epidemiology experiment, preference should be given to improved techniques for the isolation of RNA and DNA from the UCWBC.
belongs to the acyl-CoA synthetase long chain (ACSL) family of genes which encodes key enzymes in fatty acid metabolism 
is expressed in lung and thymic tissue 
. Intracellular conversion of long chain fatty acids to acyl-CoA is required for energy production. ACSL catalyzes the initial step in this conversion yielding acyl-CoAs, which are used both in the synthesis of cellular lipids and in degradation via beta-oxidation for energy production 
. Moreover, acyl-CoA synthetase can catalyze the ATP-dependent production of arachidonoyl-CoA that is a substrate for lysophosphatidyl acyltransferase involved in arachidonic acid-containing phospholipid remodeling in proliferating T cells 
. Lipid metabolism influences membrane proteins, including ion channels. In the lung, ACSL3
is found most abundantly in microsomal and cell membrane fractions, with mitochondria as the next richest source 
. Inhibition of ACS enzyme activity has been shown to cause cell death in p53
-defective lung cancer cells 
. Thus hypermethylation of this gene in T helper cells or lung tissues is expected to diminish fatty acid utilization and beta-oxidation-energy production, and possibly influence membrane phospholipid composition. Whether these functional changes directly affect asthma is unknown. However, several epidemiological studies show that fatty acid composition in milk 
, diet 
, and umbilical cord blood 
affects the development of allergy and other inflammatory diseases including asthma. For example, dietary omega-3 (n-3) fatty acids having a variety of anti-inflammatory and immune-modulating effects may affect allergy and asthma. Further mechanistic studies in in vitro
and in vivo
experimental models are needed to decipher the role of ACSL3
in childhood asthma. Interestingly, ACSL3
is located in 2q36.1 which has recently been shown to be associated with regions of the asthma susceptibility loci, in specific populations 
Finally, the current finding of a putative epigenetic marker that is associated with PAH exposure and asthma adds to other evidence from the CCCEH cohort that PAHs increase risk of respiratory symptoms and probable asthma. At age 2 years, significantly more difficulty breathing and more probable asthma were reported among children jointly exposed prenatally to PAHs and postnatally to environmental tobacco smoke (ETS) (OR for probable asthma
7.52, 95% CI 1.71–33.11, p<0.01) 
. These relationships were reexamined through age 5 years and similar trends were detected, with an observed interaction between high prenatal PAH exposure and postnatal ETS on wheeze and probable asthma at age 5 (OR
5.48, 95% CI 1.17–25.58, p<0.01) (unpublished data). A parallel cohort study in Krakow, Poland has found a significant relationship between prenatal PAH exposure and respiratory symptoms 
. In a subset of 333 subjects, PAH exposure measured during pregnancy by personal air monitors was associated with an increased risk for wheezing without cold (RR 3.8; 95% CI: 1.2–12.4), during the course of the infant's first year of life 
In conclusion, no previous studies have examined the effects of prenatal exposure to ambient air pollutants on DNA methylation patterns in genes potentially associated with the asthma phenotype of the offspring. This is an important gap in our understanding of asthma pathogenesis. Given the potential importance of epigenetic mechanisms in the etiology of childhood asthma, research is needed to identify an epigenetic profile related to PAH-associated childhood asthma and determine whether reprogramming events associated with transplacental exposure to PAHs increase risk of childhood asthma. The current report of ACSL3 as a candidate biomarker of PAH exposure and a putative predictor of PAH-associated childhood asthma represents an important first step in this area of research. Finally, the novel design of this study could serve as a prototype approach for future discoveries of additional exposure biomarkers or surrogate disease endpoints in other birth cohort studies.