磷酸三苯酯和磷酸三(1,3-二氯-2-丙基)酯对小鼠精母细胞DNA损伤和细胞周期的影响

Effects of triphenyl phosphate and tris(1,3-dichloro-2-propyl)phosphate on DNA damage and cell cycle in mouse spermatocytes

目的 探讨不同剂量的磷酸三苯酯(TPhP)和磷酸三(1,3-二氯-2-丙基)酯(TDCPP)对小鼠精母细胞DNA损伤和细胞周期的影响。方法 选取小鼠精母细胞(GC-2)为细胞模型,经TPhP和TDCPP(0、3、10、30和50μmol/L)染毒48 h后,利用CCK-8方法检测GC-2的细胞存活率,采用高内涵分析系统检测TPhP和TDCPP对细胞核碎片化程度、磷酸化组蛋白(pH2AX)、DNA同源重组修复蛋白(Rad51)及细胞周期等参数的影响。实时荧光定量PCR法分析精母细胞生长发育关键调控基因,包括环腺苷酸应答元件结合蛋白(CREB-1)、抑制素-α (inhibin-α)、粘连蛋白2(nectin-2)和增殖标记蛋白(Ki67)mRNA的表达水平。结果 与对照组相比,30和50μmol/L TPhP和TDCPP均显著降低GC-2细胞存活率(P<0.05),并引起细胞核碎片化程度加剧(P<0.01)。DNA损伤标志物pH2AX在10、30和50μmol/L TPhP组及TDCPP组均显著升高(P<0.05),Rad51蛋白在30和50μmol/L TPhP组和10、30和50μmol/L TDCPP组均显著升高(P<0.01),表明较高浓度的TPhP和TDCPP可引起DNA损伤。细胞周期分析显示,TPhP主要将细胞阻滞在G0/G1期,而TDCPP主要将细胞阻滞在G0/G1期和G2/M期。荧光定量PCR结果可见,与对照组相比,TPhP(30和50μmol/L)和TDCPP(10、30和50μmol/L)抑制精母细胞生长发育关键基因(CREB-1、inhibin-α、nectin-2和Ki67)的表达(P<0.01)。结论 TPhP和TDCPP均可引起GC-2细胞DNA损伤和细胞周期阻滞,并最终影响精母细胞的生长发育。

Objective To investigate the effects of different doses of triphenyl phosphate(TPhP)and tris(1,3-dichloro-2-pro-pyl)phosphate(TDCPP)on DNA damage and cell cycle in mouse spermatocytes.Methods Mouse spermatocyte-derived cells(GC-2)were used as the model and treated with different concentrations of TPhP and TDCPP(0,3,10,30,and 50μmol/L)for 48 hours.Cell viability was measured using Cell Counting Kit-8.The effects of TPhP and TDCPP on nuclear fragmentation,phosphoryla-ted histone H2AX(pH2AX),DNA homologous recombination repair protein(Rad51),and cell cycle parameters were analyzed using the high content imaging system.Real-time PCR was used to measure the mRNA expression levels of key genes regulating spermatocyte growth and development,including cAMP responsive element-binding protein-1(CREB-1),inhibin-α,nectin-2,and the proliferation marker Ki67.Results Compared with the control group,exposure to TPhP and TDCPP at 30 and 50μmol/L all significantly reduced GC-2 cell viability(P<0.05)and significantly accelerated nuclear fragmentation(P<0.01).The DNA damage marker pH2AX was significantly increased for TPhP and TDCPP at 10,30,and 50μmol/L(P<0.05),and Rad51 protein was significantly up-regulated for TPhP at 30 and 50μmol/L and TDCPP at 10,30,and 50μmol/L(P<0.01),indicating that higher concentrations of TPhP and TDCPP could cause DNA damage.The cell cycle analysis showed that TPhP mainly blocked the cells in the G0/G1 phase,while TD-CPP mainly blocked the cells in the G0/G1 and G2/M phases.The real-time PCR results showed that TPhP(30 and 50μmol/L)and TDCPP(10,30,and 50μmol/L)significantly inhibited the mRNA expression levels of the CREB-1,inhibin-α,nectin-2,and Ki67 genes compared with the control group(P<0.01).Conclusion Both TPhP and TDCPP can cause DNA damage and cell cycle arrest in GC-2 cells,and ultimately affect the growth and development of spermatocytes.