刘爱香, 李新, 曹晶晶, 李欢, 张志红, 梁瑞峰, 郝忠锁, 张红梅. 苯并[a]芘对人支气管上皮细胞CYP1A1GSTP1GSTM1 DNA甲基化水平和mRNA表达的影响[J]. 环境与职业医学, 2018, 35(4): 330-335. DOI: 10.13213/j.cnki.jeom.2018.17680
引用本文: 刘爱香, 李新, 曹晶晶, 李欢, 张志红, 梁瑞峰, 郝忠锁, 张红梅. 苯并[a]芘对人支气管上皮细胞CYP1A1GSTP1GSTM1 DNA甲基化水平和mRNA表达的影响[J]. 环境与职业医学, 2018, 35(4): 330-335. DOI: 10.13213/j.cnki.jeom.2018.17680
LIU Ai-xiang, LI Xin, CAO Jing-jing, LI Huan, ZHANG Zhi-hong, LIANG Rui-feng, HAO Zhong-suo, ZHANG Hong-mei. Effects of benzo[a]pyrene on DNA methylation and mRNA expression of CYP1A1, GSTP1, and GSTM1 in human bronchial epithelial cells[J]. Journal of Environmental and Occupational Medicine, 2018, 35(4): 330-335. DOI: 10.13213/j.cnki.jeom.2018.17680
Citation: LIU Ai-xiang, LI Xin, CAO Jing-jing, LI Huan, ZHANG Zhi-hong, LIANG Rui-feng, HAO Zhong-suo, ZHANG Hong-mei. Effects of benzo[a]pyrene on DNA methylation and mRNA expression of CYP1A1, GSTP1, and GSTM1 in human bronchial epithelial cells[J]. Journal of Environmental and Occupational Medicine, 2018, 35(4): 330-335. DOI: 10.13213/j.cnki.jeom.2018.17680

苯并a芘对人支气管上皮细胞CYP1A1GSTP1GSTM1 DNA甲基化水平和mRNA表达的影响

Effects of benzoapyrene on DNA methylation and mRNA expression of CYP1A1, GSTP1, and GSTM1 in human bronchial epithelial cells

  • 摘要: 目的 探讨苯并a芘(BaP)对人支气管上皮(16HBE)细胞中CYP1A1GSTP1GSTM1启动子区DNA甲基化水平和mRNA表达的影响,为BaP毒理学机制的研究提供科学基础。

    方法 体外培养16HBE细胞,取对数生长期的同一批次细胞,随机分为4组,包括1个溶剂对照组和3个BaP处理组,分别用二甲基亚砜(DMSO)及1、2、5 mmol/L BaP处理24 h,于倒置显微镜下观察细胞的形态学改变。采用CCK-8试剂盒测定细胞增殖能力,甲基化特异性PCR法检测CYP1A1GSTP1GSTM1启动子区DNA甲基化水平,实时定量PCR技术检测CYP1A1GSTP1GSTM1 mRNA表达的改变。采用SPSS 22.0软件的单因素方差分析进行比较,LSD法进行多组间的两两比较。

    结果 与溶剂对照组相比,1、2、5 mmol/L BaP处理组新生细胞数目明显增多,细胞增殖率增高,分别为(100.00±0.47)%、(125.22±0.72)%、(122.92±1.47)%、(128.44±5.97)%(P<0.05)。3个BaP组CYP1A1启动子区甲基化水平分别为8.451±1.234、8.532±0.728、11.064±0.423,低于溶剂对照组(13.917±1.094)(P<0.05)。3个BaP处理组GSTP1启动子区甲基化水平分别为4.276±0.839、3.691±0.748、2.121±0.336,也低于溶剂对照组(10.860±1.129)(P<0.05)。GSTM1启动子区甲基化水平分别为34.693±6.603、48.407±7.824、49.803±2.516;其中,1 mmol/L BaP组低于溶剂对照组(40.062±7.746),2、5 mmol/L BaP组高于溶剂对照组(均P<0.05)。3个BaP组CYP1A1 mRNA表达水平分别为0.009±0.001、0.009±0.001、0.017±0.003,低于溶剂对照组(1.067±0.064)(P<0.05);GSTP1 mRNA和GSTM1 mRNA的表达水平分别为(0.179±0.074、0.167±0.048、0.143±0.029)和(0.547±0.181、0.518±0.141、0.297±0.044),均低于溶剂对照组(0.829±0.116、0.975±0.183)(P<0.05)。

    结论 BaP对16HBE的毒作用机制可能与其细胞增殖能力增强及CYP1A1GSTP1启动子区DNA甲基化水平降低,CYP1A1GSTP1GSTM1 mRNA表达降低有关。

     

    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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