The Effect Of P, P'-DDE-indued ROS On MAPK And Mitochondria Signal Transduction Pathways

DDT,the first widely used synthetic pesticide,was given credit for having helpedone billion people live free from malaria.Its inventor was credit with the Nobel Prize.However,its bioaccumulation,long-range transport,persistence in the environmentand antiandrogenic properties raise the concern about its possible long-term adverseeffects.Though it has been restricted in using for three decades,DDT could be tracedin all humans and in far regions due to its persistence and accumulation.It has beencalculated that it would take about 10 to 20 years for DDT to disappear from anindividual if exposure would totally cease,but its metabolite DDE would possiblypersist throughout the life span.Hence,DDT was regarded as one of the 12 persistentorganic pollutants in the UNEP.p,p'-DDE,DDT's major metabolite with the highest persistence,is the form usuallyfound in human tissues in the highest concentration.It is also a kind of knownendocrine disruptor chemicals.It could play an adverse effect on the malereproductive system through direct toxicity or endocrine disrupting effect.Thoughthere have been some reports concerning p,p'-DDE's inducing apoptosis,few studiesinvestigated its effect on testicular cells.Hence,the aim of the present studies is toinvestigate p,p'-DDE's effect on the apoptosis of testicular cells.As p,p'-DDE couldinduce oxidative stress and play adverse effects on the mitochondria,we also studythe role of mitochondria and its associated protein in apoptosis induced by p,p'-DDE.The mitogen-activated protein kinases (MAPKs) are serine/threonine kinases that transduce signals from the plasma membrane to the cell nucleus.They play criticalroles in controlling cell survival,proliferation,differentiation and cellular responses tovarious harmful signals phosphorylation.Many organocholorine pesticides have beenproved to induce MAPK phosphorylation.But reports concerning p,p'-DDE's effectare still limited,particularly no details in male reproductive system.Previously,wehave examined p,p'-DDE's effect on Sertoli cell,which demonstrated that p44/42MAPK was phosphorylated in p,p'-DDE-treated Sertoli cells.In the present study,two other critical pathways,p38 and c-Jun NH2-terminal kinase (JNK),wereinvestigated.In the present study,we examined the role of lipid peroxidation,mitochondriaapoptotic pathway and MAPK signal transduction pathway in p,p'-DDE-inducedapoptosis.PartⅠEffect of p,p'-DDE on the apoptosis of Sertoli cells1,Effect of p,p'-DDE on the cellular viability of Sertoli cells antagonized bythe antioxidant NACObjective:To investigate the effects ofp,p'-DDE on the viability of Sertoli cells.Methods:Sertoli cells were treated with p,p'-DDE (10,30,50,70μmol/L) for 24 h.Inother experiment,Sertoli cells were pre-incubated with 300μM NAC for 1 h andfollowed by incubation with 50μM p,p'-DDE for 24 h.The absorbance was measuredby MTT assay.Results:The absorbance of Sertoli cells was reduced after treatment with 30,50 or 70μM p,p'-DDE (P＜0.05).In 70μM p,p'-DDE-treated group,the absorbance wasabout 50% of that in DMSO-treated group,representing the cellular viability.Whilein 50μM p,p'-DDE-treated group,the absorbance was about 70% of that inDMSO-treated group,which could be blocked by preincubation with NAC.Based onthis result,p,p'-DDE at concentration of 10,30,50μM was used in subsequentexperiments.Conclusion:p,p'-DDE could inhibit cellular viability of Sertoli cells in a dose-response relationship.NAC pretreatment could antagonize p,p'-DDE's adverseeffect.2,The effect of p,p'-DDE on SOD activity,MDA level,ROS,and mitochondrialmembrane potential antagonized by NACObjective:To investigate effects of p,p'-DDE on the SOD activity,MDA level,ROS,and mitochondrial membrane potential.Methods:The level of SOD and MDA was detected following with the protocol ofassay kit.ROS and mitochondrial membrane potential were detected by the flowcytometric analysis.Results:The SOD activity in all p,p'-DDE groups was significantly decreasedcompared to the control group (P＜0.05).The MDA level in 30 or 50μMp,p'-DDE-treated group was remarkably increased (P＜0.05).Loss of mitochondrialpotential was induced after treatment with 10,30 or 50μM p,p'-DDE (P＜0.05),suggesting the damage to the mitochondria.Preincubation with 300μMNAC for 1 hattenuated this damage significantly.50μM p,p'-DDE induced a significantincreasing level of ROS production (P＜0.05),which could be neutralized bypreincubation with NAC.Conclusion:p,p'-DDE could induce lipid peroxidation and loss of mitochondrialpotential,which could be blocked by NAC pretreatment.3,Effect of p,p'-DDE on cytochrome c,Bax,Bak,Bcl-w in the mRNA andprotein levelObjective:To investigate effects of p,p'-DDE on cytochrome c,Bax,Bak,Bcl-w inthe mRNA and protein level.Methods:Real-time PCR and Western blotting were used to detect cytochrome c,Bax,Bak,Bcl-w in the mRNA and protein level,respectively.Results:Significant increases of the steady-state cytochrome c,Bax and Baktranscript levels were found in 30 or 50μM p,p'-DDE group,which could beinhibited by antinomycin D pretreatment.No significant effect of Bcl-w was observed in the presence ofp,p'-DDE.Significant increases of Bax and Bak were observed in the protein level in Sertolicells treated with 30 or 50μM p,p'-DDE.Bcl-w protein level declined significantly in50μM p,p'-DDE group,which could be rescued by NAC.30 or 50μM p,p'-DDEtreatment induced cytochrome c translocation to cytosol.Preincubation with NACcould rescue this translocation effectively (P＜0.05).Conclusion:p,p'-DDE could affect the gene transcription and expression through theoxidative stress pathway.4,The effect of NAC on p,p'-DDE-induced phosphorylation status of p38 andJNKObjective:The effect of NAC on p,p'-DDE-induced phosphorylation status of p38and JNK.Methods:p38 and JNK phosphorylation were detected by the method of Westernblotting.Results:Significant phosphorylation of p38 was found after treatment with 30 or 50μM p,p'-DDE for 24 h,which could be inhibited by NAC preincubation.JNKphosphorylation was observed after all treatments.NAC pretreatment also inhibitedJNK phosphorylation.Conclusion:p,p'-DDE could induce MAPK phosphorylation through oxidative stress.5,The effect of NAC,MAPK inhibitors and RNA synthesis inhibitor onp,p'-DDE-induced apoptosisObjective:The effect of NAC,MAPK inhibitors and RNA synthesis inhibitor onp,p'-DDE-induced apoptosis.Methods:Sertoli cells were incubated in 50μM p,p'-DDE for 24 h.In otherexperiment,cells were pre-incubated with 300μM NAC,10μM SB202190 orSP600125,1 nM antinomycin D for 1 h followed by incubation with 50μM p,p'-DDEfor 24 h.Hoechst staining and flow cytometry were used to detect p,p'-DDE-inducedapoptosis. Results:Morphologically,Sertoli cells with 30 or 50μM p,p'-DDE exhibited specificapoptotic characters such as nuclear condensation,nuclear shrinkage andfragmentation,which could be blocked by preincubation with NAC,p38 inhibitor(SB203580) or RNA synthesis inhibitor (actinomycin D),but not with JNK inhibitor(SP600125).Conclusion:p,p'-DDE could induce apoptosis through oxidative stress-mediated p38MAPK pathway and gene transcription.PartⅡThe Effect of p,p'-DDE on apoptotic testicular cells in theweanling SD rats1,The effect of p,p'-DDE on histological changes in the seminiferous tubulesObjective:In order to investigate the effect of p,p'-DDE on histological changes inthe seminiferous tubules.Methods:22-day-old weanling SD rats were treated with p,p'-DDE via intraperitonealinjection at dose levels of 20 or 100 mg/kg body weight every other day;the controlgroup was treated with oliver oil alone.The positive control was treated with 40mg/kg flutamide.Dosing continued for 10 days.Results:HE staining indicated that the edema of interstitial tissue of testis wasaggravated in p,p'-DDE-treated groups.And the counts and layers of spermatogeniccells were obviously decreased.Conclusion:p,p'-DDE could induce histological changes in the seminiferous tubulesof the testis.2,The effect of p,p'-DDE on the apoptosis of testicular cellsObjective:To investigate the effect of p,p'-DDE on the apoptosis of testicular cells.Methods:The method of TUNEL was used to investigate the effect of p,p'-DDE onthe testicular apoptosis.Results:The apoptosis of testicular cells was induced by 20 or 100mg/kg p,p'-DDE.Conclusion:p,p'-DDE could induce apoptosis of the testicular cells.3,The effect of p,p'-DDE on the level of SOD,MDA and GSH-Px Objective:To investigate the effect of p,p'-DDE on the level of SOD,MDA andGSH-Px.Methods:The level of SOD,MDA and GSH-Px was detected following with theprotocol of assay kit.Results:The SOD activity was decreased by 20 or 100mg/kg p,p'-DDE.The MDAwas increased by 100 mg/kg p,p'-DDE.The GSH-Px level was decreased by 100mg/kg p,p'-DDE.Conclusion:p,p'-DDE could induce oxidative stress in the testis.4,The effect ofp,p'-DDE on Bax,Bak,Bcl-w in the RNA and protein levelObjective:To investigate the effect of p,p'-DDE on Bax,Bak,Bcl-w in the RNA andprotein level.Methods:Quantative real-time PCR and Western blotting were used to detect theeffect ofp,p'-DDE on Bax,Bak,Bcl-w in the RNA and protein level.Results:100mg/kg p,p'-DDE or 40mg/kg flutamide could elevated Bax and Bak inthe RNA and protein level.There was no discernable effect on Bcl-w.Conclusion:The balance between pro- and anti-apoptotic Bax-related moleculesmight play a critical role in p,p'-DDE-induced apoptosis.This study possesses some new ideas as follow:a) For the first time,we reportedthat p,p'-DDE could induce apoptosis in testicular cells throughmitochondria-mediated cellular apoptotic pathway.b) p,p'-DDE could induce p38 andJNK phoryslation through oxidative stress in Sertoli cells.But only p38 apoptoticpathway plays an important role in the apoptosis.