Objective To investigate the effects of different doses of triphenyl phosphate (TPhP) and tris(1, 3-dichloro-2-propyl)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, phosphorylated 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 TDCPP 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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