P，p'-DDE诱导ROS在线粒体和MAPK信号转导通路中的作用
摘要
DDT(Dichlorodiphenyltrichloroethane)，即1，1-(2，2，2-三氯乙叉)-双(4-氯苯)，中文名称滴滴涕，是一种在环境中持续性存在的有机污染物，是第一个被广泛使用的合成有机氯农药。它的出现给十亿人摆脱疟疾带来了福音，它的发明者M(u|¨)ller曾在1944年获得了诺贝尔医学奖。但是，它的蓄积性，长范围的迁移性，在环境中的不易降解性和内分泌干扰作用给人们带来了更多的困扰。尽管在欧美国家上世纪70年代开始，DDT就被限制使用，但是痕量DDT可在几乎所有的人群和偏远的地区中被检测到。有报道，如果一个个体不再暴露于DDT中，它的消失大概要花10-20年，但是DDT的代谢产物DDE可以终生存在。因此，DDT被列为12个持续性有机污染物(persistent organic pollutants，POPs)之一。
P，P’-DDE是DDT的最主要的代谢产物，有较高的蓄积性，因而在人体组织中的浓度也很高。它也是一种已知的环境内分泌干扰物之一，可以通过直接的毒作用和干扰生物体内正常的内分泌功能对男性生殖系统产生不良影响。很多最近的研究表明，DDT和它的代谢产物可以诱导组织或细胞发生凋亡，但是关于它们对男性生殖系统作用的报道较少。本研究的目的就是从体内和体外两个方面探讨P，P’-DDE对睾丸细胞凋亡的影响。鉴于P，P’-DDE具有脂质过氧化作用并损伤线粒体的功能，以及线粒体在细胞凋亡中的重要作用，我们还研究了线粒体及其相关蛋白在p，p’-DDE诱导细胞凋亡中的作用。
丝裂原活化蛋白激酶(mitogen-activated protein kinase，MAPK)是指可以从细胞膜转导至细胞核的酪氨酸/丝氨酸激酶，在细胞增殖、存活、分化和凋亡中起着重要的作用。很多有机氯农药已被证明具有诱导MAPK磷酸化的作用，但是对P，P’-DDE的研究较少，尤其缺乏它在男性生殖系统的研究。我们以前的研究发现P，P’-DDE可以诱导支持细胞ERK磷酸化作用。在本研究中，我们探讨了MAPK另外两个与细胞凋亡相关的通路P38和JNK的磷酸化作用在P，P’-DDE诱导支持细胞凋亡中的作用。
本研究的目的就是从体内和体外两个方面探讨P，P’-DDE对睾丸细胞的氧化应激、线粒体凋亡通路和MAPK信号转导通路的影响，并观察它对睾丸细胞凋亡的影响，进而揭示MAPK、线粒体凋亡通路、氧化应激与细胞凋亡之间的关系。
第一部分P，P’-DDE诱导睾丸支持细胞凋亡机制的研究
一、抗氧化剂NAC对P，P’-DDE抑制大鼠睾丸支持细胞活力的拮抗作用
目的：探讨P，P’-DDE对支持细胞活力的影响。
方法：对离体培养的支持细胞分别用不同终浓度的10、30、50、70μmol/LP，P’-DDE染毒24h，以及300μmol/L NAC预处理1h后再染毒50μmol/L P，P’-DDE，用MTT比色法测其在490nm的吸光度值。
结果：P，P’-DDE达到30μmol/L时，与溶剂对照组比，吸光度值显著下降，差异有统计学意义(P＜0．05)；P，P’-DDE达到50μmol/L时，细胞存活率达到70％，300μmol/LNAC预处理可以显著增加细胞存活率。
结论：P，P’-DDE可以抑制支持细胞存活力，NAC预处理可以拮抗P，P’-DDE的细胞毒作用。
二、P，P’-DDE对支持细胞总SOD活力、MDA含量、ROS、线粒体膜势能的影响，及NAC的拮抗作用
目的：检测P，P’-DDE染毒后支持细胞总SOD活力、MDA含量、ROS、线粒体膜势能的影响。
方法：用SOD和MDA试剂盒测定不同终浓度的P，P’-DDE(10、30、50μmol/L)染毒24h后，支持细胞内的总SOD活力和MDA含量。用流式细胞仪测定支持细胞内ROS和线粒体膜势能。
结果：所有剂量的P，P’-DDE可减小支持细胞总SOD活力，30、50μMP，P’-DDE增强MDA含量，差异均有统计学意义(P＜0．05)。所有剂量的P，P’-DDE可减少支持细胞线粒体膜势能，50μmol/L的P，P’-DDE可显著增加支持细胞内ROS的含量，300μmol/L NAC可以显著抑制线粒体膜势能的降低和ROS的增高，差异均有统计学意义(P＜0．05)。
结论：P，P’-DDE可以诱导氧化应激引起的线粒体功能破坏。
三、P，P’-DDE对支持细胞线粒体相关基因细胞色素c、Bax、Bak、Bcl-w表达的影响及NAC的作用
目的：检测P，P’-DDE对支持细胞细胞线粒体相关基因细胞色素c、Bax、Bak、Bcl-W表达的影响及NAC的作用。
方法：采用实时荧光定量PCR测定支持细胞中细胞色素c、Bax、Bak、Bcl-w mRNA的表达水平。采用Western blotting测定支持细胞中Bax、Bak、Bcl-w和胞浆中细胞色素c表达水平。
结果：30、50μmol/L的P，P’-DDE可显著增加支持细胞内细胞色素c、Bax、Bak mRNA表达水平，Bcl-w mRNA表达水平变化不明显，RNA合成酶抑制剂放线菌素D可以拮抗细胞细胞色素c、Bax、Bak mRNA的改变。
30、50μmol/L的P，P’-DDE可显著增加支持细胞内Bax、Bak、胞浆内细胞色素c蛋白表达水平，50μmol/L的P，P’-DDE可显著降低支持细胞内Bcl-w蛋白表达水平，NAC可以拮抗细胞色素c、Bcl-w的改变。
结论：P，P’-DDE可以影响基因转录和通过氧化应激途径影响支持细胞线粒体基因的表达。
四、P，P’-DDE对支持细胞p38和JNK磷酸化表达水平的影响及NAC的作用
目的：P，P’-DDE对支持细胞p38和JNK磷酸化表达水平的影响及NAC的作用。
方法：采用Western blotting测定支持细胞中p38和JNK蛋白磷酸化表达水平的影响。
结果：30、50μmol/Lp，P’-DDE可显著增加支持细胞p38的磷酸化，所有剂量的P，P’-DDE可显著增加支持细胞JNK的磷酸化，NAC可以拮抗p38和JNK的磷酸化改变。
结论：P，P’-DDE可以诱导氧化应激引起的MAPK磷酸化。
五、氧化应激、MAPK信号转导、基因转录在P，P’-DDE诱导支持细胞凋亡中的作用
目的：P，P’-DDE对支持细胞凋亡的影响及其与氧化应激、MAPK磷酸化、基因转录的关系。
方法：对离体培养的支持细胞分别用10、30、50μmol/L P，P’-DDE染毒24h，以及300μmol/L NAC、1 nM放线菌素D、10μmol/L SB203580及SP600125预处理1h后再染毒50μmol/L P，P’-DDE，采用Hoechst和流式细胞仪的技术，测定P，p’-DDE对支持细胞凋亡的影响。
结果：30、50μmol/L的P，P'-DDE可引起细胞核碎裂、片段化，显著诱导支持细胞凋亡，抗氧化剂NAC、p38抑制剂SB203580、RNA合成酶抑制剂放线菌素D可以拮抗P，P’-DDE引起的凋亡，而JNK抑制剂SP600125没有显著影响。
结论：P，P’-DDE可以通过氧化应激、p38和基因转录诱导支持细胞凋亡。
第二部分P，P’-DDE对断乳期SD大鼠睾丸细胞凋亡的影响
一、P，P’-DDE对断乳期SD大鼠脏器系数的比较和常规的HE染色结果
目的：不同浓度P，P’-DDE腹腔注射后大鼠脏器系数的比较和常规的HE染色结果。
方法：用不同浓度的P，P’-DDE(0、20、100mg/kg)及阳性对照氟他胺(40mg/kg)隔天对断乳期大鼠染毒10天后，处死动物，称重脏器，HE染色观察大鼠睾丸组织形态学改变。
结果：100mg/kg P，P’-DDE处理组肝脏的脏器系数和对照组相比有差异有显著性；40mg/kg氟他胺处理组前列腺、睾丸脏器系数和对照组相比有差异有显著性。HE染色结果显示对照组睾丸曲细精管及其间质结构正常、完整，支持细胞及各级生精细胞排列规则，形态正常。经DDE处理后，生精细胞排列紊乱、发生脱落，数目及层次明显减少。
结论：P，P’-DDE可以引起睾丸组织发生病理学改变。
二、P，P’-DDE对大鼠睾丸细胞凋亡的影响
目的：检测P，P’-DDE对大鼠睾丸细胞凋亡的影响。
方法：用TUNEL免疫组织化学的方法检测P，P’-DDE对大鼠睾丸细胞凋亡的影响。
结果：20、100mg/kg P，P’-DDE均可以诱导睾丸细胞凋亡，以精原细胞和精母为主，伴有支持细胞。
结论：P，P’-DDE可以诱导大鼠睾丸细胞凋亡。
三、P，P’-DDE对大鼠睾丸组织总SOD活力、MDA含量和GSH-Px活性的影响
目的：P，P’-DDE对大鼠睾丸组织总SOD活力、MDA含量和GSH-Px活性的影响。
方法：用SOD、MDA和GSH-Px试剂盒测定睾丸组织总SOD活力、MDA含量和GSH-Px活性。
结果：20、100mg/kg P，P’-DDE及40mg/kg氟他胺均诱导睾丸组织SOD活力下降，100mg/kg P，P’-DDE可以诱导睾丸组织MDA含量升高、GSH-Px活力下降。
结论：P，P’-DDE可以诱导氧化应激，引起脂质过氧化。
四、P，P’-DDE对大鼠睾丸组织Bax、Bak、Bcl-w表达的影响
目的：P，P’-DDE对大鼠睾丸组织Bax、Bak、Bcl-w mRNA表达的影响。
方法：采用实时荧光定量PCR测定睾丸组织中Bax、Bak、Bcl-w在mRNA上的表达水平；采用Western blotting测定睾丸组织中Bax、Bak、Bcl-w在蛋白上的表达水平。
结果：40mg/kg氟他胺、100mg/kg P，P’-DDE可以诱导睾丸组织Bax、Bak在RNA和蛋白水平表达升高，但对Bcl-w影响不明显。
结论：P，P’-DDE可以影响Bax家族中抗凋亡和诱凋亡基因和蛋白表达水平。
本研究的创新点有：①首次发现了P，P’-DDE可以通过激活线粒体介导的细胞凋亡通路诱导睾丸细胞发生凋亡②发现了P，P’-DDE可以通过氧化应激诱导睾丸细胞p38和JNK磷酸化，但仅仅p38凋亡通路起重要作用
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 cells
1、Effect of p,p'-DDE on the cellular viability of Sertoli cells antagonized bythe antioxidant NAC
Objective: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 NAC
Objective: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 level
Objective: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 andJNK
Objective: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 apoptosis
Objective: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 rats
1、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 cells
Objective: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 level
Objective: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.
P，P’-DDE是DDT的最主要的代谢产物，有较高的蓄积性，因而在人体组织中的浓度也很高。它也是一种已知的环境内分泌干扰物之一，可以通过直接的毒作用和干扰生物体内正常的内分泌功能对男性生殖系统产生不良影响。很多最近的研究表明，DDT和它的代谢产物可以诱导组织或细胞发生凋亡，但是关于它们对男性生殖系统作用的报道较少。本研究的目的就是从体内和体外两个方面探讨P，P’-DDE对睾丸细胞凋亡的影响。鉴于P，P’-DDE具有脂质过氧化作用并损伤线粒体的功能，以及线粒体在细胞凋亡中的重要作用，我们还研究了线粒体及其相关蛋白在p，p’-DDE诱导细胞凋亡中的作用。
丝裂原活化蛋白激酶(mitogen-activated protein kinase，MAPK)是指可以从细胞膜转导至细胞核的酪氨酸/丝氨酸激酶，在细胞增殖、存活、分化和凋亡中起着重要的作用。很多有机氯农药已被证明具有诱导MAPK磷酸化的作用，但是对P，P’-DDE的研究较少，尤其缺乏它在男性生殖系统的研究。我们以前的研究发现P，P’-DDE可以诱导支持细胞ERK磷酸化作用。在本研究中，我们探讨了MAPK另外两个与细胞凋亡相关的通路P38和JNK的磷酸化作用在P，P’-DDE诱导支持细胞凋亡中的作用。
本研究的目的就是从体内和体外两个方面探讨P，P’-DDE对睾丸细胞的氧化应激、线粒体凋亡通路和MAPK信号转导通路的影响，并观察它对睾丸细胞凋亡的影响，进而揭示MAPK、线粒体凋亡通路、氧化应激与细胞凋亡之间的关系。
第一部分P，P’-DDE诱导睾丸支持细胞凋亡机制的研究
一、抗氧化剂NAC对P，P’-DDE抑制大鼠睾丸支持细胞活力的拮抗作用
目的：探讨P，P’-DDE对支持细胞活力的影响。
方法：对离体培养的支持细胞分别用不同终浓度的10、30、50、70μmol/LP，P’-DDE染毒24h，以及300μmol/L NAC预处理1h后再染毒50μmol/L P，P’-DDE，用MTT比色法测其在490nm的吸光度值。
结果：P，P’-DDE达到30μmol/L时，与溶剂对照组比，吸光度值显著下降，差异有统计学意义(P＜0．05)；P，P’-DDE达到50μmol/L时，细胞存活率达到70％，300μmol/LNAC预处理可以显著增加细胞存活率。
结论：P，P’-DDE可以抑制支持细胞存活力，NAC预处理可以拮抗P，P’-DDE的细胞毒作用。
二、P，P’-DDE对支持细胞总SOD活力、MDA含量、ROS、线粒体膜势能的影响，及NAC的拮抗作用
目的：检测P，P’-DDE染毒后支持细胞总SOD活力、MDA含量、ROS、线粒体膜势能的影响。
方法：用SOD和MDA试剂盒测定不同终浓度的P，P’-DDE(10、30、50μmol/L)染毒24h后，支持细胞内的总SOD活力和MDA含量。用流式细胞仪测定支持细胞内ROS和线粒体膜势能。
结果：所有剂量的P，P’-DDE可减小支持细胞总SOD活力，30、50μMP，P’-DDE增强MDA含量，差异均有统计学意义(P＜0．05)。所有剂量的P，P’-DDE可减少支持细胞线粒体膜势能，50μmol/L的P，P’-DDE可显著增加支持细胞内ROS的含量，300μmol/L NAC可以显著抑制线粒体膜势能的降低和ROS的增高，差异均有统计学意义(P＜0．05)。
结论：P，P’-DDE可以诱导氧化应激引起的线粒体功能破坏。
三、P，P’-DDE对支持细胞线粒体相关基因细胞色素c、Bax、Bak、Bcl-w表达的影响及NAC的作用
目的：检测P，P’-DDE对支持细胞细胞线粒体相关基因细胞色素c、Bax、Bak、Bcl-W表达的影响及NAC的作用。
方法：采用实时荧光定量PCR测定支持细胞中细胞色素c、Bax、Bak、Bcl-w mRNA的表达水平。采用Western blotting测定支持细胞中Bax、Bak、Bcl-w和胞浆中细胞色素c表达水平。
结果：30、50μmol/L的P，P’-DDE可显著增加支持细胞内细胞色素c、Bax、Bak mRNA表达水平，Bcl-w mRNA表达水平变化不明显，RNA合成酶抑制剂放线菌素D可以拮抗细胞细胞色素c、Bax、Bak mRNA的改变。
30、50μmol/L的P，P’-DDE可显著增加支持细胞内Bax、Bak、胞浆内细胞色素c蛋白表达水平，50μmol/L的P，P’-DDE可显著降低支持细胞内Bcl-w蛋白表达水平，NAC可以拮抗细胞色素c、Bcl-w的改变。
结论：P，P’-DDE可以影响基因转录和通过氧化应激途径影响支持细胞线粒体基因的表达。
四、P，P’-DDE对支持细胞p38和JNK磷酸化表达水平的影响及NAC的作用
目的：P，P’-DDE对支持细胞p38和JNK磷酸化表达水平的影响及NAC的作用。
方法：采用Western blotting测定支持细胞中p38和JNK蛋白磷酸化表达水平的影响。
结果：30、50μmol/Lp，P’-DDE可显著增加支持细胞p38的磷酸化，所有剂量的P，P’-DDE可显著增加支持细胞JNK的磷酸化，NAC可以拮抗p38和JNK的磷酸化改变。
结论：P，P’-DDE可以诱导氧化应激引起的MAPK磷酸化。
五、氧化应激、MAPK信号转导、基因转录在P，P’-DDE诱导支持细胞凋亡中的作用
目的：P，P’-DDE对支持细胞凋亡的影响及其与氧化应激、MAPK磷酸化、基因转录的关系。
方法：对离体培养的支持细胞分别用10、30、50μmol/L P，P’-DDE染毒24h，以及300μmol/L NAC、1 nM放线菌素D、10μmol/L SB203580及SP600125预处理1h后再染毒50μmol/L P，P’-DDE，采用Hoechst和流式细胞仪的技术，测定P，p’-DDE对支持细胞凋亡的影响。
结果：30、50μmol/L的P，P'-DDE可引起细胞核碎裂、片段化，显著诱导支持细胞凋亡，抗氧化剂NAC、p38抑制剂SB203580、RNA合成酶抑制剂放线菌素D可以拮抗P，P’-DDE引起的凋亡，而JNK抑制剂SP600125没有显著影响。
结论：P，P’-DDE可以通过氧化应激、p38和基因转录诱导支持细胞凋亡。
第二部分P，P’-DDE对断乳期SD大鼠睾丸细胞凋亡的影响
一、P，P’-DDE对断乳期SD大鼠脏器系数的比较和常规的HE染色结果
目的：不同浓度P，P’-DDE腹腔注射后大鼠脏器系数的比较和常规的HE染色结果。
方法：用不同浓度的P，P’-DDE(0、20、100mg/kg)及阳性对照氟他胺(40mg/kg)隔天对断乳期大鼠染毒10天后，处死动物，称重脏器，HE染色观察大鼠睾丸组织形态学改变。
结果：100mg/kg P，P’-DDE处理组肝脏的脏器系数和对照组相比有差异有显著性；40mg/kg氟他胺处理组前列腺、睾丸脏器系数和对照组相比有差异有显著性。HE染色结果显示对照组睾丸曲细精管及其间质结构正常、完整，支持细胞及各级生精细胞排列规则，形态正常。经DDE处理后，生精细胞排列紊乱、发生脱落，数目及层次明显减少。
结论：P，P’-DDE可以引起睾丸组织发生病理学改变。
二、P，P’-DDE对大鼠睾丸细胞凋亡的影响
目的：检测P，P’-DDE对大鼠睾丸细胞凋亡的影响。
方法：用TUNEL免疫组织化学的方法检测P，P’-DDE对大鼠睾丸细胞凋亡的影响。
结果：20、100mg/kg P，P’-DDE均可以诱导睾丸细胞凋亡，以精原细胞和精母为主，伴有支持细胞。
结论：P，P’-DDE可以诱导大鼠睾丸细胞凋亡。
三、P，P’-DDE对大鼠睾丸组织总SOD活力、MDA含量和GSH-Px活性的影响
目的：P，P’-DDE对大鼠睾丸组织总SOD活力、MDA含量和GSH-Px活性的影响。
方法：用SOD、MDA和GSH-Px试剂盒测定睾丸组织总SOD活力、MDA含量和GSH-Px活性。
结果：20、100mg/kg P，P’-DDE及40mg/kg氟他胺均诱导睾丸组织SOD活力下降，100mg/kg P，P’-DDE可以诱导睾丸组织MDA含量升高、GSH-Px活力下降。
结论：P，P’-DDE可以诱导氧化应激，引起脂质过氧化。
四、P，P’-DDE对大鼠睾丸组织Bax、Bak、Bcl-w表达的影响
目的：P，P’-DDE对大鼠睾丸组织Bax、Bak、Bcl-w mRNA表达的影响。
方法：采用实时荧光定量PCR测定睾丸组织中Bax、Bak、Bcl-w在mRNA上的表达水平；采用Western blotting测定睾丸组织中Bax、Bak、Bcl-w在蛋白上的表达水平。
结果：40mg/kg氟他胺、100mg/kg P，P’-DDE可以诱导睾丸组织Bax、Bak在RNA和蛋白水平表达升高，但对Bcl-w影响不明显。
结论：P，P’-DDE可以影响Bax家族中抗凋亡和诱凋亡基因和蛋白表达水平。
本研究的创新点有：①首次发现了P，P’-DDE可以通过激活线粒体介导的细胞凋亡通路诱导睾丸细胞发生凋亡②发现了P，P’-DDE可以通过氧化应激诱导睾丸细胞p38和JNK磷酸化，但仅仅p38凋亡通路起重要作用
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 cells
1、Effect of p,p'-DDE on the cellular viability of Sertoli cells antagonized bythe antioxidant NAC
Objective: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 NAC
Objective: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 level
Objective: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 andJNK
Objective: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 apoptosis
Objective: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 rats
1、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 cells
Objective: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 level
Objective: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.
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