OBJECTIVE: Pyrosequencing was applied to study the effect of whole genome amplification (WGA) on the fidelity of DNA methylation. METHODS:EpiTect Whole Bisulfitome Kit was used to perform DNA amplification after whole genome bisulfite modification of L02,A549 and HCT116 cells. The DNA methylation of three specific genes (MGMT,GSTP1,P16) and LINE 1 sequence were analyzed by pyrosequencing. RESULTS:The DNA amplification obtained was 246 folds higher in DNA content and > 1 000 bp fragment length which met the basic requirements of DNA methylation detection. The DNA methylation level of LINE1 did not change after WGA,indicating that WGA had no effect on the whole genome methylation. However,the change of specific DNA methylation after WGA correlated with their original methylation level. When the original methylation rate was higher than 60%,the methylation condition of three genes remained stable after WGA. There was significant change with irregular trend of DNA methylation level after WGA when the initial DNA methylation ranged between ≥20%-60%. DNA methylation rate of three genes declined significantly in all cells when the initial DNA methylation level was lower than 20%. In addition,pyrosequencing result showed that the effect of WGA on genes CpG sites methylation level was similar to that on its mean methylation rate. CONCLUSION:WGA technology was suitable for the study of whole genome methylation and specific gene with higher methylation rate (≥60%). However,we must take caution for using the WGA technology when the rate of DNA methylation was relative low (< 60%).

Abstract Background: Sufficient epidemiologic evidence shows an etiologic link between polycyclic aromatic hydrocarbons (PAH) exposure and lung cancer risk. While the genetic modifications have been found in PAH-exposed population, it is unclear whether gene-specific methylation involves in the process of PAH-associated biologic consequence. Methods: Sixty-nine PAH-exposed workers and 59 control subjects were recruited. Using bisulfite sequencing, we examined the methylation status of p16INK4α promoter in peripheral blood lymphocytes (PBL) from PAH-exposed workers and in benzo(a)pyrene (BaP)-transformed human bronchial epithelial (HBE) cells. The relationships between p16INK4α methylation and the level of urinary 1-hydroxypyrene (1-OHP) or the frequency of cytokinesis block micronucleus (CBMN) were analyzed. Results: Compared with the control group, PAH-exposed workers exhibited higher levels of urinary 1-OHP (10.62 vs. 2.52 μg/L), p16INK4α methylation (7.95% vs. 1.14% for 22 “hot” CpG sites), and CBMN (7.28% vs. 2.92%) in PBLs. p16INK4α hypermethylation in PAH-exposed workers exhibited CpG site specificity. Among the 35 CpG sites we analyzed, 22 were significantly hypermethylated. These 22 hypermethylated CpG sites were positively correlated to levels of urinary 1-OHP and CBMN in PBLs. Moreover, the hypermethylation and suppression of p16 expression was also found in BaP-transformed HBER cells. Conclusion: PAH exposure induced CpG site–specific hypermethylation of p16INK4α gene. The degree of p16INK4α methylation was associated with the levels of DNA damage and internal exposure. Impact: p16INK4α hypermethylation might be an essential biomarker for the exposure to PAHs and for early diagnosis of cancer. Cancer Epidemiol Biomarkers Prev; 21(1); 182–90. ©2011 AACR.

Identification of aberrant microRNA (miRNA) expression during chemical carcinogen-induced cell transformation will lead to a better understanding of the substantial role of miRNAs in cancer development. To explore whether aberrant miRNAs expression can be used as biomarkers of chemical exposure in risk assessment of chemical carcinogenesis, we analyzed miRNA expression profiles of human bronchial epithelial cells expressing an oncogenic allele of H-Ras (HBER) at different stages of transformation induced by benzo(a)pyrene (BaP) by miRNA array. It revealed 12 miRNAs differentially expressed in HBER cells at both pretransformed and transformed stages. Differentially expressed miRNAs were confirmed in transformed cells and examined in 50 pairs of primary human non-small-cell lung cancer (NSCLC) tissues using real-time PCR. Among these miRNAs, downregulation of miR-638 was found in 68% (34/50) of NSCLC tissues. However, the expression of miR-638 in HBER cells increased upon treatment of BaP in a dose-dependent manner. The expression of miR-638 was also examined in peripheral lymphocytes from 86 polycyclic aromatic hydrocarbons (PAHs)-exposed (PE) workers. We found that the average expression level of miR-638 in peripheral lymphocytes from 86 PE workers increased by 72% compared with control group. The levels of miR-638 were correlated with the concentration of urinary 1-hydroxypyrene (1-OHP) and external levels of PAHs. Overexpression of miR-638 aggravated cell DNA damage induced by BaP, which might be mediated by suppression of breast cancer 1 (BRCA1), one of the target genes of miR-638. In summary, we suggest that miR-638 is involved in the BaP-induced carcinogenesis by targeting BRCA1.

Background: Sufficient epidemiologic evidence shows an etiologic link between polycyclic aromatic hydrocarbons (PAH) exposure and lung cancer risk. While the genetic modifications have been found in PAH-exposed population, it is unclear whether gene-specific methylation involves in the process of PAHassociated biologic consequence. Methods: Sixty-nine PAH-exposedworkers and 59 control subjects were recruited. Using bisulfite sequencing, we examined the methylation status of p16 promoter in peripheral blood lymphocytes (PBL) from PAH-exposed workers and in benzo(a)pyrene (BaP)-transformed human bronchial epithelial (HBE) cells. The relationships between p16 methylation and the level of urinary 1-hydroxypyrene (1-OHP) or the frequency of cytokinesis block micronucleus (CBMN) were analyzed. Results:Comparedwith the control group, PAH-exposedworkers exhibited higher levels of urinary 1-OHP (10.62 vs. 2.52 mg/L), p16 methylation (7.95% vs. 1.14% for 22 "hot" CpG sites), and CBMN (7.28% vs. 2.92%) inPBLs. p16hypermethylation inPAH-exposedworkers exhibitedCpGsite specificity.Among the 35 CpG sites we analyzed, 22 were significantly hypermethylated. These 22 hypermethylated CpG sites were positively correlated to levels of urinary 1-OHP and CBMN in PBLs. Moreover, the hypermethylation and suppression of p16 expression was also found in BaP-transformed HBER cells. Conclusion: PAH exposure induced CpG site–specific hypermethylation of p16 gene. The degree of p16 methylation was associated with the levels of DNA damage and internal exposure. Impact: p16 hypermethylation might be an essential biomarker for the exposure to PAHs and for early diagnosis of cancer. Cancer Epidemiol Biomarkers Prev; 1–9. 2011 AACR.