Reactive Oxygen Species-mediated Cellular Genotoxic Stress Is Involved In 1-nitropyrene-induced Trophoblast Cycle Arrest

Objectives1-Nitropyrene(1-NP),a representative nitrated polycyclic aromatic hydrocarbon(nitro-PAH)and a key component of fine particulate matter(PM2.5),is mainly derived from diesel exhaust particle.Previous study demonstrates 1-NP is a mutagen and carcinogen.International Agency for Research on Cancer(IARC)has identified 1-NP as Group 2A carcinogen in 2012.Recently,several studies have proved that 1-NP is a developmental toxicant.According to a recent report from our laboratory,maternal 1-NP exposure impaired cognitive function in adolescent pups.What is most important,maternal 1-NP exposure caused intrauterine growth restriction(IUGR)in mouse fetuses.However,the exact mechanism through which 1-NP causes fetal IUGR remains unclear.The placenta is important for sustaining fetal development.The reduction in placenta size is one of the main causes of IUGR.Our recent report observed that gestational 1-NP exposure inhibited cell proliferation in mouse placenta.Under normal circumstances,cell proliferation is promoted by cyclin-dependent kinases(CDKs)and their regulated cyclins,stimulated by exogenous and endogenous growth factors.When DNA is damaged under the external adverse factors,cellular genotoxic stress is activated.As a result,the activity of CDK/Cyclin complex is inhibited,inducing cell cycle arrest and inhibition of cell proliferation.It is well known that reactive oxygen species(ROS)is a DNA breaking agent,which induces DNA double strand breaks.As we all kown,1-NP induces excessive production of reactive oxygen species(ROS)in cells.However,whether gestational 1-NP exposure induces placental genotoxic stress remains obscure.If so,the role of cellular genotoxic stress on 1-NP-induced trophoblast proliferation inhibition needs to be further studied.Thus,the aim of this study is to investigate the role of ROS-mediated genotoxic stress in 1-NP-induced placental proliferation arrest and fetal IUGR.MethodsThis study is divided into two parts: in vitro experiments and in vivo animal experiment.The vitro experiments consist of five independent experiments.HTR8/SVneo cells were incubated with different doses of 1-NP(0,1,5,10 ?M)at different time points(12,18,24 h)in different cell experiments.Experiment 1: To investigate the effect of 1-NP on apoptosis,programmed necrosis in human placental trophoblasts,Annexin V/PI staining was used to detect apoptosis and programmed necrosis.Programmed necrotic protein RIP1,cleaved-caspase 3 and cleaved-PARP,two apoptosis-related proteins,were also detected in 1-NP-treated HTR8/SVneo cells.Experiment 2: To investigate the effect of 1-NP on the proliferation of human placental trophoblasts.CCK8 and Real-Time Cell-Analyzer DP system(RTCA)were used to detect cell viability and proliferation index,respectively.The expression level of proliferating cell nuclear antigen(PCNA)and the percentage of PCNA-positive cells were also detected in 1-NP-treated HTR8/SVneo cells.Experiment 3: To investigate the effect of 1-NP on the cell cycle in human placental trophoblasts.Flow cytometry was used to detect the cell cycle.And the expression and phosphorylation levels of cycle-related proteins were also detected in 1-NP-treated HTR8/SVneo cells.Experiment 4: To investigate the effect of 1-NP on the genotoxic stress in human placental trophoblasts.Comet assay was used to detect DNA damage.Phosphorylation level of protein kinase ataxia-telangiectasia mutated(ATM),an initiating protein of genotoxic stress,and its downstream molecule were also detected in 1-NP-treated HTR8/SVneo cells.Experiment 5: To explore the role of ROS in 1-NP-induced genotoxic stress and cell cycle arrest.The level of ROS in HTR8/SVneo cells was detected by DCFH-DA.Phenyl-N-t-butylnitrone(PBN),a free radical scavenger,was pretreated to detect the phosphorylation level of ATM.And the expression and phosphorylation levels of cycle-related proteins were also detected in 1-NP-treated HTR8/SVneo cells.In animal experiment,pregnant mice were pretreated with an antioxidant N-acetylcysteine(NAC).To explore the effects of antioxidant on 1-NP-induced placental proliferation inhibition,the weight,thickness and area of blood sinuses of placenta were measured in 1-NP-exposed mice during late pregnancy.And the expression level of PCNA was also detected in placenta of 1-NP-exposed mice.Meanwhile,the weight and length of fetal mice were also measured to explore the effects of antioxidant on 1-NP-induced fetal IUGR.ResultsIn vitro experiment,1-NP did not induce apoptosis and programmed necrosis in human placental trophoblasts.Cell valability and growth index were reduced in 1-NP-exposed placental trophoblasts.And PCNA was also downregulated in 1-NP-exposed placental trophoblasts.More than 90% of 1-NP-exposed trophoblasts were arrested in either G0/G1 or G2/M phases.CDK1 and Cyclin B,two G2/M cycle-related proteins,and CDK2,a G0/G1 cycle-related protein,were reduced in 1-NP-exposed trophoblasts.Phosphorylated retinoblastoma protein(Rb),a downstream molecule of CDK2,was inhibited in 1-NP-exposed trophoblasts.Further analysis found that DNA double-strand break was observed.?-H2 AX,another indicator of DNA double-strand break,was upregulated in 1-NP-exposed trophoblasts.Phosphorylated ATM,a key molecule of genotoxic stress,and its downstream molecule checkpoint kinase 2(Chk2)were elevated.Regulating cell division cycle 25A(Cdc25A),a downstream target of Chk2,was reduced in 1-NP-exposed trophoblasts.Pretreatment with PBN attenuated 1-NP-induced ATM phosphorylation.Moreover,pretreatment with PBN reversed 1-NP-induced Rb phosphorylation.In addition,pretreatment with PBN attenuated 1-NP-induced downregulation of cyclin B and PCNA.Finally,an in vivo experiment showed that pretreatment with NAC attenuated 1-NP-induced reduction of placenta weight and blood sinusoids area.Pretreatment with NAC attenuated 1-NP-induced downregulation of PCNA in mouse placenta.And pretreatment with NAC rescued 1-NP-induced fetal IUGR in mice.ConclusionsIn our present study,we found that 1-NP caused cell cycle arrest and genotoxic stress in human placental trophoblasts.The in vitro experiments demonstrate that ROS are involved in 1-NP-evoked genotoxic stress and cell cycle arrest.The in vivo experiments indicate that NAC,an antioxidant,alleviates 1-NP-induced placental proliferation inhibition and fetal IUGR.Our results provide experimental evidence that ROS-mediated genotoxic stress contributes partially to 1-NP-induced placental proliferation inhibition and fetal IUGR.