孕期氟他胺暴露对雄性子代小鼠生殖器官发育和氧化应激反应的影响

Effect of flutamide exposed during pregnancy on reproductive organs development and oxidative stress responsein in male mice offspring

目的探讨氟他胺染毒孕鼠后对雄性子代小鼠生殖器官发育和氧化应激反应的影响,为模型应用和机制研究进一步提供依据。方法选择7周龄ICR小鼠,受孕后雌鼠随机分为实验组和对照组,各20只,实验组孕鼠于孕12～18 d行氟他胺灌胃处理,剂量为300 mg/(kg·d);对照组以等体积大豆油灌胃。于仔鼠出生7周检查尿道发育后,每组随机选40只雄鼠检测肛门到生殖器的距离(anogential distance,AGD)、睾丸容积、阴茎长度并取部分鼠的生殖器官做组织病理学检查。随机每组取12只雄鼠的睾丸和阴茎组织,加入所需匀浆介质研磨后离心取其上清液,使用相应检测试剂盒及酶标仪检测丙二醛(malonaldehyde,MDA)含量及超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathioneperoxidase,GSH-Px)活性;实时荧光定量PCR方法检测其氧化应激基因Noxo1、Sod1及Gpx1的表达丰度。结果实验组雄性仔鼠100%出现尿道下裂(91/91),实验组与对照组相比,AGD变短[(10. 22±1. 53) vs.(17. 46±1. 25) mm],睾丸容积减小[(166. 34±26. 59) vs.(178. 25±25. 77) mm3]和阴茎变短[(7. 46±0. 76) vs.(12. 60±0. 80) mm](P <0. 05);实验组雄性仔鼠睾丸中SOD活性[(171. 08±57. 24) vs.(102. 79±15. 31) U/mg pro]及MDA[(4. 45±1. 67) vs.(2. 93±1. 00) nmol/mg pro]含量高于对照组(P <0. 05),GSH-Px[(41. 55±12. 15) vs.(78. 27±7. 60) U/mg pro]活性低于对照组(P <0. 05);实验组睾丸内Noxo1表达丰度较对照组升高(P <0. 05);睾丸内Sod1及Gpx1较对照组下降(P <0. 05);阴茎内未见SOD、MDA、GSH-Px和Sod1、Noxo1、Gpx1明显改变。结论孕鼠染毒氟他胺可致雄性子代小鼠尿道下裂和阴茎变短等发育异常、睾丸氧化/抗氧化功能异常,但对阴茎氧化/抗氧化功能无明显影响。

Objective To investigate the effects of flutamide on the reproductive organs development and oxidative stress response of male offspring after exposure to pregnant mice,and to provide further evidence for the application of the model and mechanism research.Methods Choose 7-week-old ICR mice,female mice were randomly divided into experimental group and control group after pregnancy,then flutamide was orally administered to pregnant mice with dose 300mg/(kg·d)during gestation days 12-18,while the control group was treated with equal volume of soybean oil.More than 7weeks after birth,40male mice were randomly chosen to observe the urethral development and test the anogenital distance (AGD),testicular volume and penile length,and then some of these mice's reproductive organs were took for histopathological examination.After grind and centrifuge to obtain the supernatant,twelve penis and testes were randomly selected for examining the level of malondialdehyde (MDA),superoxide dismutase (SOD ),and glutathioneperoxidase (GSH-Px)use corresponding kit and microplate reader.The abundance of oxidative stress related genes Noxol,Sodl and Gpxl were detected by qPCR.Results The incidence of hypospadias in male mice in the treatment group was 100%(91/91).AGD in the treatment group was (10.22±1.53)mm,which was significantly lower than the control(17.46±1.25)mm (P <0.05).The volume of testicles in the treatment group was (166.34±26.59)vs. (178.25±25.77)mm3in the control group (P <0.05).The length of the penis in the treatment group was (7.46±0.76)vs.(12.60±0.80)mm in the control group (P < 0.05).In the testicles,the level of SOD was(171.08±57.24)vs.(102.79±15.31)U/ mg pro,MDA was(4.45±1.67)vs.(2.93±1.00)nmol/mg pro,GSH-Px was (41.55 ±12.15)vs.(78.27±7.60)U/rag pro (P <0.05).The abundance of Noxol in the testis from the treatment group was higher than that in the control group,while the expressions of Sodl and Gpxl in testis from the treatment group were lower than those in ,the control group(P <0.05).There is no significant difference in SOD,MDA,GSH-Px levels and Sodl,Noxol,and Gpxlexpressions were found in the penises of both groups. Conclusion Prenatal exposure of flutamide may cause developmental abnormalities such as hypospadias and shortening of the penis in male offspring,and abnormal testicular function of oxidation and anti-oxidation.However,there is no significant effect on penis oxidative/anti-oxidant function