Effects of triphenyl phosphate and tris(1,3-dichloro-2-propyl)phosphate on DNA damage and cell cycle in mouse spermatocytes
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 50 μmol/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 50 μmol/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 50 μmol/L(P<0.05),and Rad51 protein was significantly up-regulated for TPhP at 30 and 50 μmol/L and TDCPP at 10,30,and 50 μmol/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 50 μmol/L)and TDCPP(10,30,and 50 μmol/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.
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