| Nitropolycyclic aromatic hydrocarbons(NitroPAHs)are derivatives of polycyclic aromatic hydrocarbons(PAHs)with at least one nitro functional group(-N02)on the aromatic ring,which are a kind of important environmental pollutants.1-Nitropyrene(1-NP)is the representative substance of NitroPAHs,and WHO have identified 1-NP as a marker of NitroPAHs exposure.Sufficient evidences shown that NitroPAHs are carcinogenic,mutagenic,cytotoxic,developmental toxic and reproductive endocrine toxic,but the mechanisms of NitroPAHs-induced male reproductive endocrine damage have not been elucidated.This study explored the effects of 1-NP exposure on testosterone synthesis.We further explored the role of GCN2-eIF2a signal in 1-NP-induced testosterone synthesis dysfunction.We evaluated the role of reactive oxygen species(ROS)and mitochondrial dysfunction in 1-NP-induced testosterone synthesis dysfunction.We studied the protective effects of GCN2 silencing,GCN2 kinase activity inhibition,and mitochondrial targeting antioxidant MitoQ on 1-NP-induced testosterone synthesis dysfunction.We then explored the role of ROS mediated GCN2 activation in 1-NP-induced testosterone synthesis dysfunction.This study provides an experimental evidence for elucidating the molecular mechanisms of 1-NP-induced testosterone synthesis dysfunctionPart 1:The effect of 1-nitropyrene exposure on testosterone levels in miceObjective The aim of this study is to investigate the effects of 1-NP exposure on testosterone levels in miceMethods We carried out a long-term in vivo experiment to explore the effects of subchronic 1-NP exposure on testosterone levels in mice.24 adult male mice were divided into control group and 1-NP group randomly,each group consists of 12 mice Mice in the 1-NP group were given 1 mg/kg of 1-NP by gavage per day for 70 consecutive days.Mice in the control group were given blank solvent.Mice were sacrificed after 24 hours of the last 1-NP treatment.Whole blood,testes and epididymis were collected.Radioimmunoassay was used to measure serum and testicular testosterone levels.The left epididymis was dissected to count the number of sperm in epididymis.The TUNEL analysis was used to detect the apoptotic cells in seminiferous epithelium.The number of testicular Ley dig cells was counted by immunohistochemistry of 33HSD.In order to clarify the effects of single 1-NP exposure on testosterone levels in mice,we implemented the time dependent and dose dependent 1-NP exposure experiments in mice.In the dose dependent experiment,50 adult male mice were divided into 5 groups(0 mg/kg,0.625 mg/kg,1.25 mg/kg,2.5 mg/kg and 5 mg/kg)randomly.Mice in the treatment groups were given 1-NP,and mice in the control group were given blank solvent.Mice were sacrificed after 24 hours of 1-NP treatment.In the time dependent experiment,we selected 4 time points(0 h,6 h,24 h and 72 h),and 40 adult male mice were divided into 4 groups randomly.The whole blood and testis of mice were collected.The serum and testicular testosterone levels were measured by radioimmunoassayResults Subchronic 1-NP exposure did not affected spermatogenesis in mouse testes Subchronic 1-NP exposure did not reduced the number of Leydig cells in mouse testes Both subchronic 1-NP exposure and single-dose 1-NP exposure down-regulated serum testosterone levels and testicular testosterone levels significantly in mice,and single-dose 1-NP exposure reduced serum testosterone levels and testicular testosterone levels in a dose-dependent and time-dependent mannerConclusion Both subchronic 1-NP exposure and single-dose 1-NP exposure can reduce serum testosterone levels and testicular testosterone levels in mice.Subchronic 1-NP exposure can't reduces the number of Ley dig cells in mouse testes,suggesting that 1-NP exposure may reduce testosterone levels by inhibiting the ablity of testosterone synthesis in Ley dig cells.However,the specific molecular mechanism of 1-NP exposure-induced testosterone synthesis dysfunction in mouse testes is still unclearPart 2:Role of ROS-mediated GCN2-eIF2? signal activation in 1-Nitropyrene-induced steroidogenesis dysfunction in MiceObjective The aim of this study is to investigate the effects of 1-NP exposure on the mRNA expressions and protein levels of testosterone synthases in mouse testes,and explore the effects of 1-NP exposure on the GCN2-eIF2? signaling pathway in mouse testes.We will preliminarily explore the role of ROS-mediated GCN2-eIF2? signaling activation in 1-NP-induced testosterone synthesis dysfunctionMethods This study is consisted of 6 experiments.In experiment 1,in order to study the effects of single-dose 1-NP exposure on the mRNA expressions and protein levels of testosterone synthases,we conducted in vivo experiments and TM3 cell experiments We explored the effects of single 1-NP exposure on protein levels of testosterone synthases in mouse testes.In the dose dependent experiment,50 adult male mice were divided into 5 groups(0 mg/kg,0.625 mg/kg,1.25 mg/kg,2.5 mg/kg and 5 mg/kg)randomly.Mice in the treatment groups were given 1-NP,and mice in the control group were given blank solvent.Mice were sacrificed after 24 hours of 1-NP treatment.In the time dependent experiment,we selected 4 time points(0 h,6 h,24 h and 72 h),and 40 adult male mice were divided into 4 groups randomly.The mice were sacrificed and mouse testes were collected.The mRNA expressions of testosterone synthases in mouse testes was detected by RT-qPCR.The protein levels of testosterone synthases in mouse testes were detected by Western blotting.We explored the effects of 1-NP exposure on protein levels of testosterone synthases in TM3 cells.In the dose-dependent experiment,TM3 cells were treated with differ concentrations of 1-NP(0 ?M,0.625 ?M,2.5 ?M and 10 ?M)under LH stimulation.After 1-NP treatment for 24 hours,the TM3 cells were collected.The mRNA expressions of testosterone synthases in TM3 cells were detected by RT-qPCR.The protein levels of testosterone synthases in TM3 cells were detected by Western blottingIn experiment 2,we conducted mouse experiments and TM3 cell experiments to investigate the effects of 1-NP exposure on GCN2-eIF2? signal.We explored the effects of single 1-NP exposure on GCN2-eIF2? signal in mouse testes.In the dose dependent experiment,50 adult male mice were divided into 5 groups(0 mg/kg,0.625 mg/kg,1.25 mg/kg,2.5 mg/kg and 5 mg/kg)randomly.Mice in the treatment groups were given 1-NP,and mice in the control group were given blank solvent.Mice were sacrificed after 24 hours of 1-NP treatment.In the time dependent experiment,we selected 4 time points(0 h,6 h,24 h and 72 h),and 40 adult male mice were divided into 4 groups randomly.The mice were sacrificed and mouse testes were collected.The protein levels of GCN2-eIF2? signal-related molecules in mouse testes were detected by Western blotting.We explored the effects of 1-NP exposure on GCN2-eIF2? signal in TM3 cells In the dose-dependent experiment,TM3 cells were treated with differ concentrations of 1-NP(0 ?M,0.625 ?M,2.5 ?M and 10 ?M).After 1-NP treatment for 24 hours,the TM3 cells were collected.In the time-dependent experiment,the differ time points(0 h,6 h,12 h and 24 h)were selected after a single treatment of 1-NP at 10?M.The TM3 cells were treated at differ time points and collected at the same time point.The protein levels of GCN2-eIF2? signaling-related molecules in TM3 cells were detected by Western blottingIn experiment 3,in order to study the role of GCN2-eIF2? signaling activation in the 1-NP-induced testosterone synthesis dysfunction in TM3 cells,the GCN2's kinase activity inhibitor GCN2iB and GCN2 targeting small interfering RNA(siGCN2)were used.On one hand,we investigated the protective effects of GCN2 kinase inhibition on 1-NP-activated GCN2-eIF2? signal and 1-NP-decreased protein levels of testosterone synthases in TM3 cells.Firstly,TM3 cells were incubated with GCN2iB at 50 nM for 1 h to inhibit GCN2 kinase activity,and then TM3 cells were treated with 1-NP at 10 ?M for 24 hours.After,TM3 cells were collected.On the other hand,we investigated the protective effects of GCN2 silencing on 1-NP-activated GCN2-eIF2? signal and 1-NP-decreased protein levels of testosterone synthases in TM3 cells.Firstly,TM3 cells were incubated with siGCN2 for 6 hours to silence GCN2,and then TM3 cells were treated with 1-NP at 10 ?M for 24 hours.After,TM3 cells were collected.The protein levels of testosterone synthases and GCN2-eIF2? signal-related molecules were detected by Western blottingIn experiment 4,we explored the effects of 1-NP exposure on mitochondrial dysfunction in TM3 cells.In the time dependent experiment,we selected 4 time points(0 h,6 h,12 h and 24 h),the TM3 cells were treated with 1-NP at 10 ?M.The mitochondrial potential and level of mitochondrial derived ROS(mtROS)in TM3 cells were measured at the same time point.In the dose dependent experiment,TM3 cells were treated with 1-NP at differ concentrations(0 ?M,0.625 ?M,2.5 ?M and 10 ?M)The mitochondrial membrane potential,mtROS,ATP content,and OXPHOS protein levels in TM3 cells were measured at 24 hours after 1-NP treatment.The MitoSOX staining was used to detect the level of mtROS in TM3 cells.The JC-1 staining was used to measure the mitochondrial membrane potential in TM3 cells.The ATP contents were detected by the luciferase-luciferin method in TM3 cells.The protein levels of OXPHOS subunits in TM3 cells were detected by Western blottingIn experiment 5,we explored the effects of 1-NP exposure on the production of ROS and oxidative damage in TM3 cells.In the time dependent experiment,we selected 4 time points(0 h,6 h,12 h and 24 h),and TM3 cells were treated with 1-NP at 10 ?M ROS levels and oxidative damage related molecules in TM3 cells were measured.In the dose dependent experiment,TM3 cells were treated with 1-NP at differ concentrations(0 ?M,0.625 ?M,2.5 ?M and 10 ?M),the ROS levels and oxidative damage related molecules in TM3 cells were measured at 24 hours after 1-NP treatment.The DCFH-DA staining was used to detect the level of ROS in TM3 cells.The Western blotting was used to detect the protein levels of NOX4 and HO-1 in TM3 cellsIn experiment 6,in order to explore the protective effect of MitoQ pretreatment on 1-NP exposure-induced testosterone synthesis dysfunction and GCN2-eIF2? signal activation,we conducted mouse experiments and TM3 cell experiments.In TM3 cell experiments,TM3 cells were incubated with MitoQ at 1 ?M for 1 hour,and then TM3 cells were treated with 1-NP at 10 ?M for 24 hours.After,TM3 cells were collected to detect the protein levels of testosterone synthases and GCN2-eIF2? signal-related molecules using Western blotting.The DCFH-DA staining was used to detect the level of ROS in TM3 cells.The MitoSOX staining was used to detect the levels of mtROS in TM3 cells.The ATP contents were detected by the luciferase-luciferin method in TM3 cells.In mouse experiments,we set 4 groups,including control group,MitoQ group,1-NP group and 1-NP+MitoQ group.Two doses of MitoQ(4 mg/kg)were given by intraperitoneal injection at 2 h and 24 h before 1-NP treatment.1-NP was given at 1.25 mg/kg by gavage.Mice were sacrificed at 24 h after 1-NP treatment.Serum and testicular testosterone were measured by radioimmunoassay.The protein levels of testosterone synthases and GCN2-eIF2a signal-related molecules in mouse testes were detected by Western blottingResults In mouse experiments,single-dose 1-NP exposure down-regulated the protein levels of testosterone synthases in a dose-dependent and time-dependent manner in mouse testes.The TM3 cell experiments showed that,1-NP exposure reduced the protein levels of testosterone synthases in a dose-dependent in a dose dependent manner Single 1-NP exposure activated the GCN2-eIF2? signal and upregulated the protein level of HSP60 in mouse testes and TM3 cells,in a dose-dependent and time-dependent manner.1-NP exposure increased the levels of mtROS and intracellular ROS,increased the protein levels of NOX4 and HO-1,and decreased the mitochondrial membrane potential in a dose-dependent and time-dependent manner in TM3 cells.We also observed that 1-NP exposure down-regulated the ATP content and protein levels of OXPHOS subunits,including C V-ATP5A,CIV-MTCO1,C ?-SDHB and C ?-NDUFB8,in a dose-dependent manner in TM3 cells.It is noteworthy that,MitoQ pretreatment,GCN2 silence and inhibition of GCN2 kinase activity,alleviated the GCN2-eIF2? signal activation induced by 1-NP exposure and restored the 1-nitroyrene decreased-protein level of testosterone synthases.Moreover,MitoQ pretreatment alleviated the 1-NP-increased mtROS and intracellular ROS,and restored the decreased ATP contents induced by 1-NP in TM3 cells.In addition,MitoQ pretreatment alleviated 1-NP induced-activation of GCN2-eIF2? signal and restored 1-NP decreased protein levels of testosterone synthases in the mouse testis.Importantly,MitoQ pretreatment alleviated the 1-NP-decreased serum and testicular testosterone levels in miceConclusion This study found that 1-NP exposure can reduces the protein levels of testosterone synthases in mouse testes and TM3 cells.1-NP exposure activates the GCN2-eIF2? signal in mouse testes and TM3 cells.1-NP can induces testosterone synthesis dysfunction through GCN2-eIF2? singal pathway.The mitochondrial derived reactive oxygen species plays a key role in the mitochondrial dysfunction,the activation of GCN2-eIF2? signal and the decreased protein levels of testosterone synthases,which all are induced by 1-NP exposure.This study provides an experimental evidence that the ROS mediated GCN2-eIF2? signal activation contributes to 1-NP-induced testosterone synthesis dysfunctionTaken together,we can draw the following conclusions based on the above results Both sub chronic 1-NP exposure and single-dose 1-NP exposure can reduce serum testosterone levels and testicular testosterone levels in mice.1-NP exposure can reduces the protein levels of testosterone synthases in mouse testes and TM3 cells.1-NP can induces testosterone synthesis dysfunction through ROS mediated GCN2-eIF2? singal pathway. |
PDF Full Text Request