PAH, Epigenetic and Asthma Figure 3. Real-time PCR analysis of gene expressions of ACSL3 in non-small cell lung cancer H1299 cell line in response to (A) 5-aza- deoxycytidine (5AZA-dC) and (B) benzo[a]pyrene (BaP) and bisulfite genomic sequencing analysis of ACSL3 promoter methylation status in response to BaP (C). A: Cells were treated with 0.5 and 1.0 mM 5-AZA-dC with DMSO as control every 2 days for a total of 8 days. B: Cells were treated with 0.01, 0.1 and 1.0 nM BaP with DMSO as control every 2 days for a total of 4 days. RNA was isolated, reverse transcribed and underwent real-time PCR. The 2-DDCt method was used to calculate the relative expression level of transcripts normalized to b-actin. *Statistically significant differences between exposed and control was accepted at p,0.05. C: Diagram represents methylation status of ACSL3 promoter of H1299 cells exposed to BaP assayed by bisulfite genomic sequencing. Cells were treated with 0.01, 0.1 and 1.0 nM BaP with DMSO as control every 2 days for a total of 4 days. Open circle: unmethylated CG; closed circle: methylated CG. Putative transcription factor binding sites such as Sp1, AP2, GCF, c-fos and junB are shown in scale with the particular CG sites on the promoter. doi:10.1371/journal.pone.0004488.g003 children were classified as ‘‘asthmatic’’ or ‘‘nonasthmatic.’’ (‘‘with or without a parental report of asthma’’). Study Sample Selection All subjects with stored UCWBC and paired FPT were eligible for inclusion in this proof-of-principle study. The population was dichotomized on the median PAH value of the entire cohort 3 (2.3 ng/m ). Half of the 56 subjects in the present study were selected from among the ones above this median (the high PAH exposure group) and half from among those below the cohort median (the lower PAH exposure group). Twenty subjects (10 high and 10 low exposure) were used to identify UCWBC DNA sequences whose methylation status was dependent on maternal PAH exposure (high versus low) and to validate concordant transcript changes in matched FPT samples. All 56 samples were then used to validate the association between PAH exposure and ACSL3 59CGI methylation and to explore the association between ACSL3 59CGI methylation and childhood asthma status. Table 6 shows the characteristics of the cohort population and study sample, respectively. The sample was representative of the cohort population in terms of maternal age, child’s sex, median PAH exposure, and percentage of children with probable asthma; however, the sample differed with respect to ethnicity. Table 7 showed the PAH distribution and variation in PAH levels in selected and unselected cohorts. Monitoring and Sample Collection Personal prenatal air monitoring was conducted for two consecutive days on all participants during the 3rd trimester of pregnancy as previously described [4–6]. Levels of pyrene and eight carcinogenic PAHs were analyzed (4). Total PAH levels for each participating mother were computed as the sum of the eight carcinogenic PAHs, where the values below the limit of detection were imputed as 0.125 ng/m3 (the median value between 0 and the limit of detection). Umbilical cord blood (30–60 mL) was collected at delivery. The buffy coat, packed red blood cells, and plasma samples were separated and stored at 280uC. DNA (100 ng–500 ng) was extracted from UCWBCs. For some UCWBC samples, matched FPTs were also available and total RNA was extracted using a TRIZOLH (Invitrogen, CA) according to manufacturer’s protocol. All samples were coded. Methylation Sensitive Restriction Fingerprinting (MSRF) MSRF was performed in a single-blinded manner on asthma classification, as previously described [19,20]. Five hundred ng of UCWBC DNA was restriction-digested using MseI [New England Biolabs, MA] alone or in combination with BstUI [New England Biolabs, MA] and amplified by PCR with various combinations of paired arbitrary primers (Table 8). Following electrophoresis, candidate sequences displaying differential methylation status between the ‘‘high PAH group’’ and ‘‘low PAH group’’ were isolated, extracted from the gel, cloned, identified and aligned with various databases. Sequences matched to known genes and with 59-CGIs were selected for bisulfite genomic sequencing and confirmation studies of their regulatory function in gene expression. Six candidates [ACSL3, RAD21, DUSP22, SFMBT2, SCD5 and WWOX] were selected. Treatment of H1299 with 5-aza-dC and BaP H1299 cell line is a gift from Dr. Carolyn M. Klinge (University of Louisville) and were maintained in RPMI-1640 medium (Invitrogen, CA) supplemented with 10% fetal bovine serum (Hyclone, UT), 10 mM HEPES (Invitrogen, CA), 1 mM sodium pyruvate (Invitrogen, CA) and 4500 mg/L glucose (Invitrogen, CA). For demethylation assays, cells were treated with 0.5 and 1.0 mM 5-aza-dC (Tocris, MO) with DMSO (Sigma, MO) as control every 2 days for a total of 8 days. For BaP treatment, cells were exposed to 0.01, 0.1 and 1.0 nM BaP (Sigma, MO) with DMSO as control every 2 days for a total of 4 days. Medium was replenished with new drug of 5-AZA-dC or BaP every 48 hr. DMSO was added in the control samples. RNA was isolated, reverse transcribed and undergone real-time PCR. DNA was isolated, bisulfite-modified and undergone bisulfite genomic sequencing. Bisulfite genomic sequencing Genomic DNA (100 ng) from UCWBC samples or H1299 cells was modified with sodium bisulfite by using the EZ Methylation Modification Kit (Zymo Research, CA) before conducting PCRs. In silico analyses and detailed database searches were used to predict the 59-CGI(s) in each gene. Primers were designed to amplify ,0.9 to 1.4 kb fragments encompassing the 59-CGI(s) of a candidate gene from bisulfite-modified DNA (Table 8). Amplicons were generated from individual UCWBC sample and subcloned into the pCR2.1 vector (Invitrogen, CA); eight or more clones were picked and sequenced (Macrogen, Korea). MSPCR analyses Bisulfite-modified genomic DNA was subjected to MSPCR analyses [27,28]. Three sets of primers were designed to amplify regions (Figure 3A; R1, R2, and R3) within the 59-CGI that exhibited the highest degree of differential methylation between the high and low PAH group. For each region, the M primer set amplifies the target sequence when it is methylated; the U primer set only amplifies the unmethylated allele (Table 8). Thirty-five- cycle PCRs were performed with 1 unit Platinum Taq DNA polymerase (Invitrogen, CA) using the following conditions: denature at 94uC for 30 s, anneal at 58uC for 1-min, and extend at 72uC for 1-min, followed by 12-min final extension. PCR products were separated on 2% agarose gel and visualized with ethidium bromide. Real-time reverse-transcriptase-PCR (real time RT-PCR) Total RNA was isolated from FPT samples matched to UCWBC samples or from H1299 cells as previously reported [14,16]. Steady state mRNA levels of the genes under investigation PLoS ONE | www.plosone.org 9 February 2009 | Volume 4 | Issue 2 | e4488