Abstract:
Objective To investigate promoter DNA methylation and mRNA expression levels of CYP1A1, GSTP1, and GSTM1 in human bronchial epithelial (16HBE) cells exposed to benzoapyrene (BaP), and provide scientific evidence for toxicological mechanism of BaP.
Methods Well-grown 16HBE cells in the exponential growth from the same batch in vitro were randomly divided into four groups treated with dimethyl sulfoxide (DMSO) (solvent control group) and 1, 2, or 5 mmol/L BaP (BaP groups), respectively. Following BaP treatment for 24 h, we observed the morphological changes under an inverted microscope, determined cell proliferation using CCK-8 kit, detected promoter DNA methylation in CYP1A1, GSTP1, and GSTM1 using methylation-specific PCR method, and measured mRNA expression levels of CYP1A1, GSTP1, and GSTM1 using real-time quantitative PCR. Comparisons among groups were made using ANOVA in SPSS 22.0 software, and pairwise comparisons were analyzed by LSD.
Results The numbers of newborn cells of the three BaP groups were increased compared to the solvent control group, and the cell viabilities were (125.22±0.72)%, (122.92±1.47)%, and (128.44±5.97)% in the 1, 2, and 5 mmol/L BaP groups, respectively, which were significantly increased compared to the solvent control group(100.00±0.47)% (P < 0.05). The methylation levels of CYP1A1 promoter were 8.451±1.234, 8.532±0.728, and 11.064±0.423 in the three BaP groups, respectively, lower than that of the solvent control group (13.917±1.094) (P < 0.05). The methylation levels of GSTP1 promoter were 4.276±0.839, 3.691±0.748, and 2.121±0.336 in the three BaP groups, respectively, also lower than that of the solvent control group (10.860±1.129) (P < 0.05). The methylation levels of GSTM1 promoter were 34.693±6.603, 48.407±7.824, and 49.803±2.516 in the three BaP groups, respectively; the 1mmol/L BaP group showed a lower methylation level than the solvent control group (40.062±7.746), but the 2 and 5 mmol/L BaP groups showed higher methylation levels than the solvent control group (P < 0.05). The mRNA expression levels of CYP1A1 were 0.009±0.001, 0.009±0.001, and 0.017±0.003 in the BaP groups, respectively, which were significantly decreased compared to the solvent control group (1.067±0.064) (P < 0.05). The mRNA expression levels of GSTP1 were 0.179±0.074, 0.167±0.048, and 0.143±0.029 in the BaP groups, respectively, significantly decreased compared to the solvent control group (0.829±0.116) (P < 0.05). The mRNA expression levels of GSTM1 were 0.547±0.181, 0.518±0.141, and 0.297±0.044 in the BaP groups, respectively, significantly decreased compared to the solvent control group (0.975±0.183) (P < 0.05).
Conclusion The toxicological mechanism of BaP may be related to its enhanced cell proliferation, decreased DNA methylation in the promoter regions of CYP1A1 and GSTP1, and reduced expression of CYP1A1, GSTP1, GSTM1 mRNA in 16HBE.
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