【作者】 张英彪；

【导师】 吴成秋；

【作者基本信息】 南华大学 ， 卫生毒理学， 2008， 硕士

【摘要】 目的:观察苯与甲醛对雄性小鼠生殖系统的联合毒作用,探讨二者联合作用类型和相关毒作用机制,为进一步全面评价苯和甲醛的雄性生殖毒性提供实验及理论依据。方法:昆明种雄性小鼠108只,按3×3析因设计随机分为9个染毒组,每组12只,甲醛染毒剂量为（0、5、20 mg/kg）、苯染毒剂量为（0、100、400mg/kg）;甲醛用生理盐水稀释,苯用植物油稀释,连续灌胃染毒5天后,再饲养35天处死动物,观察染毒和饲养期间小鼠的生长发育、体重增长情况,计算睾丸脏器系数;取出附睾观察精子死亡率和畸形率;每组6只鼠睾丸做常规病理学切片、HE染色,观察睾丸的形态结构学变化;另6只鼠睾丸匀浆离心,取上清液用分光光度法检测一氧化氮（Nitrogen monoxidum;NO）和钙(Calcium;Ca²⁺)含量、总抗氧化能力（Total Antioxidant Capacity;T-AOC）、Na⁺-K⁺-ATP、Ca²⁺-Mg²⁺-ATP酶活性。结果:1.染毒期间各组小鼠的毛发均无光泽,活动减少,进食差,在高剂量联合组约50%小鼠有排稀便现象;在饲养期间对照组的毛发光泽、活动和食欲在5天内就完全恢复正常,而各染毒组恢复缓慢,尤其是高剂量组和联合染毒组在处死前仍未见恢复。2.饲养期间各染毒组小鼠体重比对照组增长缓慢,与对照组比较差异有显著性（P<0.01）,染毒剂量越高,体重增长抑制越明显;小鼠睾丸脏器系数随染毒剂量增加而降低,低苯组与对照组无差异（P>0.05）,其他各组与对照组比较差异有显著性（P<0.01）,并存在一定的量-效关系。苯和甲醛对小鼠体重增长抑制呈相加效应（P>0.05）,对小鼠睾丸脏器系数的影响表现为有协同效应（P<0.01）。3.对照组和各染毒组均有死精子和精子畸形现象,对照组精子死亡率和畸形率分别为45.17‰、58.33‰,染毒组精子的死亡率和畸形率高于对照组（P<0.01）,苯与甲醛染毒剂量越高,精子的死亡率和畸形率越高;死精子细胞膜不完整,HE染色有红染;精子畸形表现主要有:大头精子、小头精子、香蕉头精子、勾形头精子、双头精子、短尾精子、折尾精子等;甲醛和苯对小鼠精子的损害表现为协同效应。4.睾丸病理组织学观察显示:对照组曲细精管形态规则,边缘完整,曲细精管管壁内精原母细胞、初级精母细胞和次级精母细胞由外至内排列紧密,层次分明清晰,曲细精管管腔位于中央处,管腔内可见大量初步发育成熟的精细胞,无变性损害。单毒物和毒物联合染毒组可见曲细精管形态不规则,边缘极不完整,大小不一,曲细精管管壁内的细胞由外至内排列不紧密,精原母细胞、初级精母细胞和次级精母细胞有些变性损害,甚至有细胞缺失的现象,层次不清晰,曲细精管管腔偏位,甚至没有管腔,部分管腔内隐约可见少量发育成熟的精子,有些管腔内未见成熟精子。染毒浓度越高,病理损害越明显。5. T-AOC在各染毒组随染毒剂量增加而减低,与对照组比较差异均有高度统计学意义（P<0.01）,并呈现明显的量-效关系。甲醛与苯对小鼠睾丸T-AOC的抑制作用表现为协同效应。6.各组小鼠接触不同剂量的甲醛和苯后,睾丸中Na+-K+-ATP、Ca²⁺-Mg²⁺-ATP酶活性随染毒剂量增加而降低,与对照组比较差异有显著性（P<0.01）;苯和甲醛之间有交互作用,对Na+-K+-ATP、Ca²⁺-Mg²⁺-ATPase的抑制作用表现协同作用。7.各染毒组小鼠睾丸细胞的NO和Ca²⁺含量随染毒剂量的增加而增高,与对照组比较差异有显著性（P<0.01）;苯与甲醛对小鼠睾丸细胞NO和Ca²⁺含量的影响也是协同作用。结论:1.苯和甲醛可抑制小鼠的生长,损伤小鼠的睾丸生殖细胞,使精子的死亡率和畸形率增加,二者的联合毒作用表现为协同效应。2.苯和甲醛对小鼠睾丸生殖细胞的损伤可能是通过降低睾丸生殖细胞的抗氧化能力、抑制ATP酶活性、影响第二信使（钙离子失衡）等方式造成睾丸组织和精子的损伤。更多还原

【Abstract】 Objective:To study the reproductive toxicity of benzene and formaldehyde, and to explore the joint toxic action type and possible mechanism of benzene and formaldehyde for reproductive system in male mouse; to provide experimental and theoretical evidence for further evaluating the reproductive toxicity of benzene and formaldehyde.Method:108 male Kunming mice were randomly divided into nine groups according to 3×3 factorial design, 12 male mice in each group. The lavage dose of formaldehyde was 0, 5, 20mg/kg body weight respectively, and the lavage dose of benzene was 0, 100, 400 mg/kg body weight respectively. Formaldehyde was diluted with physiologic saline, and benzene was diluted with vegetable oil. Formaldehyde and benzene was administered by intragastric administration for continuous five days, once a day and 0.1ml/10g body weight. All mice were fed 35 days after lavage. The growing and development situation of mice were observed during the period of exposure to poison and feeding. After feeding 35 days, all mice were killed, and testicular organ coefficient was calculated. The sperm mortality and malformation rate were observed in all mice epididymis. In each group, the testes of six mouce were used to doing conventional pathology section, HE staining to observe the testicular tissue morphous changes; the others were homogenised and centrifuged, the concentration of NO, T-AOC and Ca²⁺, the activity of Na⁺-K⁺-ATP and Ca²⁺-Mg²⁺-ATP were detected in the supernatant using spectrophotometric method.Results:1. During the period of exposure to benzene and formaldehyde, the hair of mice in treatment groups and control group had no luster, all mice were less activity, poor appetite, and the stool of 50% mice in high dose group was watery. During the period of feeding, the hair, activity and appetite of mice in the control group were recovered to normal within 5 days, but which in treatment groups recovered more slowly, especially, which in high-dose group and the joint group could not recover until ante mortem.2. During the period of feeding, the weight increases of mice in treatment groups were more slowly than that of control group, and the difference was statistical significance（P<0.01）. The higher the dose of poisons was, the more evident growth inhibition was, and the joint toxic effect of benzene and formaldehyde on body weight was synergism. The testicular organ coefficient decreased with the increasing of lavage dose, and the coefficient in treatment groups was lower than that of in control group（P<0.01）,there was statistical significance except the low-dose benzene group, and the inhibition of Benzene and formaldehyde showed dose-effect relationship. The synergistic toxic effect of benzene and formaldehyde was found in testicular organ coefficient.3. There were dead sperm and the sperm shape abnormality in the control and the treatment groups. In control group, the percentages of dead sperm and malformed sperm were 45.17‰, 58.33‰respectively. and there was higher in the treatment groups compared with the control group（P<0.01）. The larger the dose of benzene and formaldehyde were, the higher the percentages of dead sperm and malformed sperm were, and the synergistic toxic effect of benzene and formaldehyde could been detected. The cell membrane of the dead sperm was not intact, and HE stain showed change red to dye。The main type of sperm malformation were big head sperm, small head sperm, double head sperm, banana head sperm,crook head sperm,short tail sperm and fold tail sperm etc.4. The testicular histopathology in control group showed: the seminiferous tubules had regular morphology and clear border; and spermatocyte, primary spermatocyte, secondary spermatocyte arranged densely form outer to inner, and their shape and structure were clear in convoluted seminiferous tubule; the convoluted seminiferous tubule was situated in the cernter, and it contained large numbers of mature and normal sperm cells. The testicular histopathology in the treatment groups showed: the seminiferous tubules had irregular morphology and unclear border, and spermatocyte, primary spermatocyte, secondary spermatocyte didn’t arrange closely from outer to inner, and their shape and structure were damaged in convoluted seminiferous tubule. The convoluted seminiferous tubule was deviated form the center, even it was lack, only parts of seminiferous tubules contained some mature sperm cells.5. T-AOC in the treatment groups was decreased with exposure doses increasing, there was statistical significance compared with the control group（P<0.01）,and there was dose-effect relationship. A synergistic effect on T-AOC was showed evidently in the joint exposure groups of benzene and formaldehyde.6. With the increases of the exposure doses of benzene and formaldehyde, ATP enzyme activity in the treatment groups was decreased （P<0.01）. Benzene and formaldehyde had a significant synergistic toxic effect on Na⁺-K⁺-ATP、Ca²⁺-Mg²⁺-ATP enzyme activity.7. With the increases of the exposure doses of benzene and formaldehyde, the concentrations of NO and Ca²⁺ in the treatment groups were increased （P<0.01）. Benzene and formaldehyde had a significant synergistic toxic effect on the concentrations of NO and Ca²⁺.Conclusion:1. Formaldehyde and benzene can inhibited the growth and development of mice, and mice reproductive cells can be damaged by them, the percentages of dead sperm and malformed sperm were increased. Formaldehyde and benzene have synergistic toxic effect on reproductive system in male mouse.2. The possible mechanism of Formaldehyde and Benzene on reproductive toxicity in male mice is to damage antioxygen ability, to inhibit ATP enzyme activity and to influence NO metabolism and Ca²⁺ disbalance in generative cell of testiculus.更多还原