organogenesis resulting in serious ailments in later life. The best studied mechanisms underpinning the latter scenario is the induction of aberrant DNA methylation of regulatory sequences in susceptible genes by environmental toxicants during critical developmental periods, leading to inappropriate gene expression and disease pathogenesis in later life [12–16]. This body of research suggests that transplacental exposure to high levels of airborne PAHs could cause aberrant DNA methylation changes, leading to dysregulation of gene expression and perhaps childhood asthma. These alterations could occur, not only in target organs/ tissues such as fetal lung or the immune system [11], but also in peripheral tissues such as fetal circulating white blood cells and placental tissues, thus providing opportunities to discover and develop highly sensitive and specific, minimally invasive biomark- ers for exposure assessments or clinical indications [17]. The primary objective of this study was to explore whether transplacental exposure to PAHs in humans induces epigenetic reprogramming involving aberrant DNA methylation of specific genes that might be mechanistically related to childhood asthma or airway inflammation. Study subjects were children born to nonsmoking Dominican and African American women living in Northern Manhattan and the South Bronx. They were partici- pants in the Columbia Center for Children’s Environmental Health (CCCEH) cohort study previously described [4–6]. Mothers completed prenatal air monitoring and donated a maternal and/or umbilical cord blood sample and, in some cases, fetal placental tissue (FPT) at delivery. Umbilical cord white blood cells (UCWBC) and matched FPT from 20 cohort subjects were used initially for candidate identification and selection, as it is not feasible to obtain lung/immune cells in newborns. UCWBC provide a reasonable surrogate for our target organ/tissue (lung/ immune cells) because they contain stem cells that can populate the lungs in later life and also provide a rich source of T cells [18] which are important producers of cytokines and other asthma mediators. The matched FPT provides ample tissue for obtaining fetal RNA to verify correlative changes in gene expression. A larger sample of 56 cohort subjects was used to validate the markers identified. Methylation of a 59-CpG island (CGI) in acyl- CoA synthetase long-chain family member 3 (ACSL3) was found to be positively and significantly associated with the level of maternal PAH exposure and with a parental report of asthma symptoms prior to age 5 in this exploratory study. Results Identification of candidate genes and selecting the most relevant candidate Figure 1 shows the work flow used in the discovery of candidates and strategies employed for selecting candidate genes associated with prenatal airborne PAH exposure and with asthma. Twenty participants were randomly selected without knowledge of asthma classification: ten with maternal PAH exposure levels below the CCCEH cohort median of 2.3 ng/m3 (‘‘low PAH group’’) and ten with prenatal PAH levels above the cohort median (‘‘high PAH group’’). Methylation-sensitive restriction fingerprint- ing [19,20] was performed to identify candidate sequences displaying differential methylation status between high and low PAH groups. Both hypo- and hyper-methylated candidate sequenc- es in the high PAH group when compared to the low PAH group were selected. In silico analyses revealed that 19 out of 31 sequenced candidates were homologous to known genes (Table 1) and 6 of these aligned to a CGI(s) located in the 59 flanking region (proximal promoter and/or exon/intron 1) of a known gene (59-CGI) (Figure 2). These 6 genes therefore were chosen as candidates for PAH, Epigenetic and Asthma further investigation. Unexpectedly, all six candidates were found be hypermethylated sequences in the high PAH group when compared to the low PAH group. A literature search showed that they are expressed in the lung and/or lymphoid tissues and have functions related to inflammatory/or other immune responses (dual specificity phosphatase 22; DUSP22, accession # BC022847, [21]), DNA damage and repair (RAD21 homolog (S. pombe); RAD21, accession # BC050381, [22]), fatty acid metabolism (ACSL3, accession # BC041692, [23]; stearoyl-CoA desaturase 5; SCD5, accession # NM_001037582, [24] ), gene transcription (Scm-like with four mbt domains 2; SFMBT2, accession # NM_001029880, [25]; WW domain containing oxidoreductase; WWOX, accession # AF211943, [26] ). Bisulfite sequencing analyses of the initial 20 UCWBC samples confirmed a good agreement between hypermethylation of all of the candidates’ 59-CGI(s) and either high or low PAH exposure status, consistent with MSRF findings (Table 1). We next determined the strength of the relationship between the percent of methylation of a candidate gene’s 59-CGI in UCWBC DNA samples and its transcript expression level in matched FPT RNA samples (Table 2 and 3). The degree of methylation of a region encompassing the entire predicted 59-CGI for each candidate gene was determined by bisulfite sequencing (Figure 2, the location of the amplicon was marked as BSPCR) and mRNA levels determined by real time RT-PCR. The percentage of promoter methylation (Met%) determined by bisulfite sequencing and relative gene expression levels (RER) determined by real time RT-PCR of all six candidates tested on 14 paired UCWBC DNA and FPT RNA samples are presented in Table 2. The degree of concordance between these two parameters for each candidate is shown in Table 3. We found that the degree of ACSL3 59-CGI methylation and the level ACSL3 transcript expression exhibited the expected inverse relationship with the highest concordance and significance (t = 20.45; p,0.01) when compared to the other candidates. These findings indicate that the ACSL3 59-CGI is likely a regulatory CGI since increased cytosine methylation in this sequence in UCWBC DNA samples was found to be significantly associated with transcriptional silencing of the gene in matched FPTs. To further demonstrate that the methylation status of the ACSL3 promoter affects gene expression, we used a lung non-small carcinoma cell line H1299 as a model system. Two series of experiments were performed. In the first series, we treated H1299 cells with a DNA demethylation agent, 5-aza-29-deoxycytidine (5- aza-dC), and found that ACSL3 expression was significantly increased (Figure 3A) along with promoter demethylation (data not shown). These data provide evidence that demethylation of the ACSL3 promoter is associated with enhancement of gene expression. In the second series of experiments, H1299 cells exposed to low doses of benzo[a]pyrene (BaP; 0.01 to 1 nM), a representative PAH, showed a dose-dependent decrease in ACSL3 transcript expression as indicated by real time RT-PCR analysis (Figure 3B) and increased promoter hypermethylation as revealed by bisulfite sequencing (Figure 3C). These data strongly support the notion that the CpG island in the ACSL3 promoter plays a regulatory role in gene expression. Association of methylation status of ACSL3 59CGI with PAH exposure in the larger sample ACSL3 was chosen for subsequent assessment of the relation- ships between prenatal PAH exposure and gene 59-CGI methylation status and for exploratory analyses of PAH exposure status and asthma classification in a larger sample of 56 children from the CCCEH cohort. The organization of the 59-flanking sequence in the promoter region of ACSL3 is shown in Figure 4A. Before we conducted the full PLoS ONE | www.plosone.org 2 February 2009 | Volume 4 | Issue 2 | e4488