【作者】 王兴庄；

【导师】 徐世文；

【作者基本信息】 东北农业大学 ， 临床兽医学， 2008， 硕士

【摘要】 硝基苯(Nitrobenzene,NB)是应用广泛的化工基础原料,也是一种毒性较强的物质,主要用于染料、医药、农药及炸药等行业,美国环保局己将其列入优先控制的污染物名单中。已有研究表明,硝基苯对哺乳动物造血、肝、肾及中枢神经系统可造成损伤,而对其生殖功能损伤的报道较少。本试验以雄性昆明小鼠为研究对象,灌胃染毒,设置三个剂量组:低剂量(26 mg/kg体重)、中剂量(52 mg/kg体重)、高剂量(105 mg/kg体重),每个剂量组设相应的空白对照和溶剂对照。其中低剂量、高剂量染毒时间为30 d,中剂量染毒时间为10 d、20 d、30 d。通过硝基苯染毒对睾丸和副性腺的毒性、对睾酮(T)、促卵泡激素(FSH)及生化指标、抗氧化功能、细胞凋亡、生殖细胞DNA损伤、Bcl-2、Bax mRNA的表达、雄激素受体(AR)表达的检测,探讨了硝基苯对雄性小鼠生殖毒性的机理。研究结果表明:1.通过血清生化指标的检测,表明硝基苯可以导致血清中碱性磷酸酶(ALP)、琥珀酸脱氢酶(SDH)、r-谷胺酰转移酶(r-GT)、乳酸脱氢酶(LDH)活性降低,引起相应生理功能的变化并导致生精功能的障碍。2.应用放免法和免疫组织化学法分别检测血清内T、FSH的含量和AR表达水平,证实硝基苯可以导致血清中T含量降低,FSH含量升高,说明间质细胞和支持细胞受损;而AR表达降低,削弱了AR对T转录的调节作用。影响T和FSH对生殖系统的调节功能,导致生殖系统内分泌紊乱。3.试验结果证实硝基苯能导致睾丸和血清超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活性下降,总抗氧化能力(T-AOC)下降,丙二醛(MDA)含量升高,表明硝基苯可引起睾丸抗氧化功能下降,诱发氧化应激,揭示氧化损伤是硝基苯致小鼠雄性生殖毒性的机制之一。4.通过病理组织学观察、DNA Ladder、单细胞凝胶电泳(SCGE)和原位末端标记(TUNEL)法检测,说明硝基苯能够导致生殖细胞凋亡,进而引起小鼠生殖系统发生损伤。5.应用RT-PCR法检测生殖细胞Bcl-2、Bax mRNA表达水平,表明硝基苯干扰Bcl-2、Bax mRNA的表达,Bcl-2/Bax的比值明显降低,是硝基苯导致生精细胞凋亡的机制之一。综上所述,硝基苯可以抑制小鼠生殖功能,破坏抗氧化系统,诱导氧化应激,干扰Bcl-2、Bax mRNA,促使生殖细胞凋亡增加,进而发挥硝基苯生殖毒性作用。本实验从器官、细胞、亚细胞和分子水平较系统地研究了硝基苯对雄性小鼠生殖系统的毒性作用,进一步探讨了硝基苯致雄性生殖机能障碍的毒理机制,丰富了毒理学知识,并为评价硝基苯的雄性生殖毒性提供科学依据。更多还原

【Abstract】 Nitrobenzene(NB) is used extensively in dye, medicine, pesticide, dynamite and so on as basic raw material in chemical engineering. It is also a material with a high toxicity. United States Environmental Protection Agency has put it in pollutant list of priority controlling. Some studies indicated that main harm of NB was liver, kidney and central nervous system. The report is still few about the effect of NB on reproductive system of the male mice. In this test, male KM mice were divided into three dose groups(low dose(26 mg/kg), middle dose(52 mg/kg),and high dose(105 mg/kg)) and each dose group respectively included natural control group and solvent control group. Low dose group and high dose group used intragastric administration lasting 30 days, and middle dose respectively lasting 10days, 20days and 30days. After those days, those mice were killed to detected toxicity of NB to testicle and glandula accessoria, T and FSH content and biochemical indicator change, antioxidative function, apoptosis, germ cell DNA damage, the expression of Bcl-2、Bax mRNA in germ cell and AR expression in order to explore the reproductive toxicity mechanism induced by NB. The result showed as follows:1. Detection of biochemical indicator in serum showed that NB induced decrease of ALP, SDH, r-GT and LDH activity and resulted in dysspermatogenesis.2. The content of T, FSH and the expression level of AR were respectively detected by radioimmunity assay and immunohistochemical method. The result showed that NB leaded to the decrease of T content and the increase of FSH content, which revealed damage of interstitial cell and Sertoli’s cell. NB induced the decrease of AR expression level, which weakened regulation of AR to T transcription. These findings suggested that NB affected regulation of reproductive system and resulted in endocrine disturbance of reproductive system.3. The detection of antioxidative function revealed that the activity of SOD, CAT, GSH-Px and T-AOC were decreased and the content of MDA was increased in testis and serum. This showed that NB induced oxidative stress by the decrease of antioxidative function and this might be one of the maleness reproductive toxicity mechanism induced by NB.4. Apoptosis was detected by pathohistology observation, DNA Ladder, SCGE and TUNEL. The result showed that NB induced germ cell apoptosis and resulted in damage of genital system in male mice.5. The expression level of Bcl-2 and Bax mRNA was detected by RT-PCR. Bcl-2/Bax ratio was decreased obviously and this might be an important mechanism of NB inducing apoptosis.Above all, NB inhibited reproductive function of the male mice, destroyed antioxidative function, induced oxidative stress. In addition, NB interfered with Bcl-2、Bax mRNA expression level, and induced germ cell apoptosis. All these results showed reproductive toxicity of NB.In this study, reproductive toxicity of the male mice induced by NB was revealed from organic, cellular, subcellular and molecular level, and its toxicology mechanism was explained. These supplemented toxicology knowledge and provided evidence for evaluating male reproductive toxicity induced by NB.更多还原