苯并(a)芘染毒致人支气管上皮细胞的周期改变及BPDE-DNA加合物形成

目的:通过观察苯并(a)芘(BaP)染毒致人支气管上皮细胞16HBE细胞周期改变及BPDE-DNA加合物形成的情况,探讨DNA损伤与细胞周期阻滞之间的关系。方法:用不同浓度BaP(0、1、2、4、8、16、32 μmol/L)染毒16HBE细胞24 h,用16 μmol/L BaP染毒16HBE细胞不同时间(0、1、2、4、8、12、24 h)以检测BaP染毒16HBE细胞的剂量和时间效应。再根据上述结果选择16 μmol/L BaP染毒16HBE细胞4 h后,恢复不同时间(0、1、2、4、8、12、24 h),处理结束后采用流式细胞术检测细胞周期分布情况,酶联免疫法和荧光免疫组化法检测BPDE-DNA加合物表达。结果:与正常对照组相比,随着BaP染毒浓度和染毒时间的增加,S期细胞所占比例均增加(P<0.05或P<0.01)。16 μmol/L BaP染毒16HBE细胞4 h后,恢复早期(4~12 h)S期细胞所占比例与恢复0 h相比明显增加(P<0.05),恢复24 h时S期细胞所占比例(24.52%)与恢复0 h相比明显降低(P<0.01),而与正常对照组(26.41%)相比差异无统计学意义(P>0.05)。随着染毒浓度增加和染毒时间延长,16HBE细胞内BPDE-DNA加合物含量逐渐增加,与正常对照组相比差异均有统计学意义(P<0.01),染毒后恢复1 h时BPDE-DNA加合物含量与恢复0 h组相比显著增加(P<0.01),而后随恢复时间延长逐渐下降。回归分析显示BaP染毒后细胞S期所占比例与BPDE-DNA加合物含量符合Cubic方程(R²=0.386,P=0.01)。结论:BaP染毒所致BPDE-DNA加合物的形成与S期阻滞密切相关。

Changes of cell cycle and BPDE-DNA adducts in human bronchial epithelial cells exposed to benzo(a)pyrene

OBJECTIVE:To explore the correlations of BPDE-DNA adducts and cell cycle in human bronchial epithelial cells (16HBE) exposed to benzo(a)pyrene. METHODS:16HBE cells were exposed to BaP at concentrations of 0, 1, 2, 4, 8, 16 and 32 μmol/L for 24 hours. 16HBE cells exposed to 16 μmol/L BaP were collected at different times points (0, 1, 2, 4, 8, 12 and 24 h), and cells exposed to 16 μmol/L for 4 h were collected after different recovery times (0, 1, 2, 4, 8, 12 and 24 h). Flow cytometry was used to evaluate the cell cycle. BPDE-DNA adducts were evaluated by enzyme linked immunosorbent assay (ELISA) and fluorescent immunohistochemistry method. RESULTS:Compared with the control group, the proportion of S phase cells were significantly increased with increasing concentration and exposure time (P<0.05 or P<0.01). The proportion of S phase cells of 16HBE increased at early recovery (4-12 h), but they underwent a significantly reduction with increasing recovery time. Compared with the normal control (26.41%), there was not a significantly difference at 24 h recovery (24.52%). Compared with the normal control, the levels of BPDE-DNA adducts were significantly elevated with increasing concentration and exposure time (P<0.01). Compared with the recovery a t 0 h, the adducts were significantly inereased at 1 h recovery (P<0.01), and then the level of adducts decreased gradually with increasing recovery time. Regression analysis showed that the proportions of S phase cells and BPDE-DNA adduct levels conformed to the Cubic equation (R²=0.386, P=0.01). CONCLUSION: There was a close correlation between S phase arrest and levels of BPDE-DNA adduct induced by BaP.