The samples included 50 asthmatic subjects and 50 control subjects who were recruited from the Children’s Hospital of Nanjing Medical University and the Affiliated hospital of Nanjing university of Traditional Chinese Medicine from 2020-2021, respectively. Asthmatic subjects were diagnosed by a clinician. The control subjects were with no history of inflammatory disease and atopy. The Nanjing Medical University Clinical Research Ethics Committee, Nanjing, China, reviewed and approved the protocols of this study. Written informed consent was obtained from the participants’ parent for the use of samples in this study. Whole blood samples from each asthmatic or control subjects were collected. The samples were centrifuged at 3000 rpm for 10 min. The supernatant was stored at -20 °C until analysis. White blood cell DNA was isolated by using TIANamp Genomic DNA Kit (TIANGEN, DP304-03, China) following the instructions from the manufacturer. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood using Lymphocyte-Human Cell Separation Media (Cedarlane, Southern Ontario, Canada).
2.2 Total IgE and IL-17A analysis
The determination of total IgE and IL-17A levels in children serum were performed using Human IgE ELISA Kit (AMEKO, Shanghai, China) and Human IL-17A ELISA Kit (Cusabio, Wuhan, China) according to the manufacturer’s instructions, respectively. The absorbance was measured at a wavelength of 450nm (Infinite M2000, Tecan Trading AG, Switzerland).
2.3 PAHs analysis by GC-MS
Briefly, 0.2 mL of the serum was spiked with internal standards（D10-Phe, D12-Chr, Accustandard, New Haven, CT, USA）. Then 0.5 mL of 6mol/L hydrochloric acid, 0.5 mL of isopropanol and 3mLof n-hexane/methyl tertiary butyl ether (v/v,1:1) were added and vortexed for 2 min. After centrifugation at 5000 rpm for 10 min, the organic phase was collected. The above extraction procedure was carried out for three times. The mixed extract was evaporated under gentle nitrogen, and then was redissolved in 100 μl of n- hexane and prepared for quantification.
The quantitative analysis of target PAH compounds in the serum, including fluorene (Flu), phenanthrene (Phe), anthracene (Ant), fluoranthene (Fla), pyrene (Pyr), benzo(a)anthracene (BaA), chrysene(Chr), benzo(b)fluoranthene (BbF), benzo(k)fluoranthene(BkF), benzo(a)pyrene (BaP), indeno(1,2,3-cd)pyrene (InP), and dibenzo(a,h)anthracene (DBA), were performed using gas chromatograph mass spectrometer (TRACE 1310, Thermo Fisher Scientific, USA) with a chromatographic column (DB-5MS, 30 m, liquid film thickness 0.25 μm, internal diameter 0.25 mm). The program of the GC analysis was described in our previous work . The quantification was performed using standard curves by a standard mixture solution (Supelco Company, 20 μg/mL). Isotopically labeled internal standards were used with the recoveries of 97.0% for D10-Phe, and 123.7% for D12-Chr. Human reference serum (RS10-100-4, BETHYL) was as blank. Low molecular weight PAH isomers (naphthalene, acenaphthylene, acenaphthene) had poor recoveries due to their high volatility. So, they were excluded in the analyte. Limits of detection (LODs) were defined as a signal-to-noise ratio of 3:1. If the concentration was below LOD, it was reported as not detected (ND) and assigned a concentration of zero. INP and DBA were not well separated. Benzo[ghi]perylene was also exclude as the concentrations in a large proportion of samples were below LOD.
2.4 The analysis of one carbon metabolites by UPLC-MC
The serum was pretreated according to a previous study . For metabolite quantitation, isotopically labeled one carbon metabolites including S-adenosylhomocysteine-d4(SAH-d4), L-Methionine-d4 (Met-d4) (Tornoto Research Chemicals, North York, Canada) were used as internal standards. In brief, 200μL of serum was added to 1mL of methanol containing 100μg/mL ascorbic acid, 100μg/mL citric acid and 1.5mg/mL dithiothreitol (DTT). The internal standard solution was spiked at 200ng/ml concentration. After vortexed for 2min and centrifuged at 13000rpm for 15min at 4℃, the supernatant was dried under nitrogen at room temperature. The residue was reconstituted with 100μL of methanol/water (3:1, v/v) containing 10μg/mL of ascorbic acid, citric acid, and DTT, and stored at −20 ℃ for further analysis. The calibration curve standards, 5-methyltetrahydrofolate (5-MT), serine (Ser), glycine (Gly), methionine (Met), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), homocysteine (Hcy), betaine (Betaine) (Sigma Aldrich, St. Louis, MO, USA), were prepared by spiking the internal standard solutions. Metabolites were quantified using UPLC Ultimate 3000 system (Dionex, Germering, Germany) with an Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The separation of the samples was performed on a Waters ACQUITY BEH-C18 column (2.1mm×100mm, 1.7μm) at a flow rate of 0.3 mL/min. Mobile phase A was water containing 20mM ammonium formate and 0.15% (v/v) formic acid, and mobile phase B was methanol containing 0.15% (v/v) formic acid. The column temperature was at 35±1°C. The injection volume of samples was 20μL. A linear gradient procedure was described in Table S1. The effluent was unsplitted. Mass spectrometric analyses in the positive ion mode in full scan MS/SIM mode and the parameters were given in Table S2. The temperature of the turbo ion electrospray was set at 320 °C. The ion spray voltage was 3200V. Metabolite concentrations were calculated from their peak area ratios and the calibration curve. Surrogate standards were used with the recoveries of 74.04% for SAH-d4 and 93.91 for Met-d4. Human reference serum (RS10-100-4, BETHYL) was as blank. Limits of detection (LOD) were defined as a signal-to-noise ratio of 3:1. If the concentration was below LOD, it was reported as not detected (ND) and assigned a concentration of zero.
2.5 Bisulfite sequencing PCR
DNA methylation status of the LINE-1 (X58075.1) was detected by bisulfite genomic sequencing PCR amplification (BSP). In silico analyses and detailed databases searches were used to predict the 5'-CpG islands in LINE-1 gene. For LINE-1, BSPCR primers were designed to amplify a CpG-rich region spanning from 113bp to 357 bp from the transcription start site, which contains 15 CpG sites, and the full length is 275bp. BSPCR primer sequences were 5'-TTATTAGGGAGTGTTAGATAGTGGG-3' for forward; 5'-CCTCTAAACCAAATATAAAATATAATCTC -3' for reverse. 200 ng of genomic DNA was used for bisulfite treatment using the EZDNA Methylation™ Kit (Zymo Research, CA, USA). The bisulfite treated DNA was amplified with methylation specific primers using GoTaq Green Master Mix (Promega, WI, USA) and optimized PCR condition (95°C for 10 min, 35 cycles of 95°C for 30 sec, 52.6°C for 1 min and 72°C for 2 min, followed by an extension at 72°C for 10 minutes. PCR products were purified by Gel Extraction Kit (E.Z.N.A., USA) and subcloned into pMD 19-T Vector (TaKaRa, Japan). Ten clones from each sample were sequenced (TsingKe Biological Technology, China) to obtain direct measures of DNA methylation at each CpG site in the promoter region. Sequencing data was analyzed with the DNAMAN to examine the methylation status. The percentage of DNA methylation was calculated with the formula: methylated CG / (methylated CG+ unmethylated CG) *100%.
2.6 ChIP-qPCR assay for H3K4me3 enrichments
Chromatin immunoprecipitation (ChIP) was conducted with a ChIP-IT Express Enzymatic (Active Motif) according to the manufacturer’s protocol. Homogenate from zebrafish larvae was fixed with 1% formaldehyde for 10 min at room temperature to cross-link proteins and DNA, and then add Glycine Stop-Fix Solution (1ml 10× Glycine Buffer,1ml 10× PBS and 8ml distilled H2O) rocking at room temperature for 5 minutes to stop cross-linking. After washing with 1× PBS at room temperature three times, the tissue was pelleted by centrifugation for 10 min at 2,500 rpm at 4℃ then resuspended in ice-cold Lysis Buffer supplemented with protease inhibitor cocktail and 100mM PMSF. Transfer the cells to an ice-cold dounce homogenizer. Dounce on ice with 10 strokes to aid in nuclei release and centrifuge for 10 min at 5,000 rpm in a 4℃ microcentrifuge to pellet the nuclei. Add the working stock of Enzymatic Shearing Cocktail (200U/ml) and incubate at 37℃ for 15 minutes and add ice-cold 0.5M EDTA to stop the reaction. Centrifuge for 10 min at 15,000 rpm in a 4℃ microcentrifuge. The supernatants were immunoprecipitated with H3K4me3 (1:50, #9751; Cell Signaling Technology) antibody with rotation after taking out part of as input DNA, which was followed by incubation with protein G magnetic beads for 4 h at 4 °C. The anti-IgG (1:1000, #3900; Cell Signaling Technology) was used as negative control. Protein G magnetic beads antibody/chromatin complexes were collected, washed, and eluted. Then, cross-links were reversed, and DNA was purified and analyzed via real-time PCR. The ChIP qPCR primer sequences were as follows: 5’-CTAGTTCTCATCACTCTCTACTCCC-3’ (forward) and 5’-ATTGAATTTAACAATTCTTTTGTTG-3’ (reverse), -738bp to -512 bp from transcription start site , and β-actin was used as an internal reference . The levels of bound DNA sequences were then calculated using the percent input method (2- [Ct (ChIP)‑ Ct (Input)] × 100) by calculating the qPCR signal relative to the input sample.
2.7 Statistical analyses
The differences between asthmatic children and the control were analyzed using student’s t test by GraphPad Prism 8 software (GraphPad Software, La Jolla, CA). Differences were considered statistically significant at P＜0.05.The association between the PAHs level and asthma was determined with logistic regression. Age and gender were considered as covariates in the regression model. Pearson correlation was used to assess the associations between PAHs and one carbon metabolites and methylation. A redundancy analysis (RDA) was performed to determine the multivariate relationship between PAHs and sample distribution and one carbon metabolites by R package. In addition, mediation analysis for the association between PAHs and asthma mediated by intermediates was implemented considering one carbon metabolites and DNA methylation and histone methylation as mediators with reference to our previous work .