邻苯二甲酸二（2-乙基己基）酯及氯氰菊酯致大鼠性腺发育障碍的研究

Study on the mechanism of di-2-ethylhexyl phthalate and cypermethrin inducing gonadal dysgenesis in the prepubertal male rats

【摘要】目的探讨邻苯二甲酸二（2-乙基己基）酯（di_2．ethylhexylphthalate，DEHP）和氯氰菊酯（cypermethrin，CYP）单独及联合染毒引致青春前期雄性大鼠性腺发育不良的作用机制。方法SPF级3周龄雄性sD大鼠40只，随机分为对照组（玉米油）、DEHP单独染毒组（500mg／kg）、CYP单独染毒组（80mg／kg）及联合染毒组（DEH500mg／kg＋CYP80ms／ks），各组10只，每天灌胃1次，连续30d。于末次染毒24h后处死动物，测定大鼠体重及睾丸湿重，并计算睾丸系数；放射免疫法测定血清睾酮水平；电子显微镜观察睾丸生殖细胞超微结构变化；实时荧光定量PCR法测定卵泡刺激素受体（FSHR）、雄激素结合蛋白（ABP）、抑制素B（INHB）、波形蛋白（VIM）的基因表达水平；蛋白免疫印迹法（Westernblot）测定FSHR、ABP、INHB及VIM的蛋白表达水平，利用2x2析因方差分析，对比各组间的差异，通过交互图判断DEHP与CYP的交互作用。结果与对照组比较，DEHP、CYP单独及联合染毒组睾丸重量分别下降39．3％．59．2％，睾丸系数分别下降19．7％--58．6％，血清睾酮水平均分别显著下降49．1％～62．7％，差异均有统计学意义（P〈0．05或P〈0．01）。各染毒组睾丸生殖细胞超微结构均可见不同程度的病变。与对照组比较，DEHP单独染毒组FSHR、ABP、INHB、VIMmRNA表达水平分别下调1．72，2．64，2．83及1．79倍，蛋白表达分别下调65．2％，53．7％，70．1％及51．9％，差异均有统计学意义（P〈0．05或P〈0．01）；CYP单独染毒组ABP、INHB的mRNA表达水平分别下调1．72及2．06倍，蛋白表达分别下调38．3％及49．7％，差异有统计学意义（P〈0．05）；联合染毒组FSHR、ABP、INHB及VIM的mRNA表达水平分别下调1.62、2．00、2．35及1．54倍，FSHR，INHB及VIM蛋白表达分别下调52．1％，53．9％及58．8％，差异均有统计学意义（P〈0．05）。经2x2析因方差分析，DEHP、CYP联合染毒对ABP、INH的交互作用有统计学意义（P〈0．05），交互作用呈拮抗效应。结论DEHP、CYP单独及联合染毒均可致青春前期大鼠性腺发育不良，二者均可不同程度的干扰睾丸支持细胞功能相关的基因与蛋白的表达，联合染毒呈一定拮抗作用，两者相比，以DEHP的生殖毒性更强。

Objective Toinvestigatethemechanismofdi-2-ethylhexylphthalate （DEHP） andcypermethrin （CYP） inducing gonadal dysgenesis in prepubertal male rats. Methods A total of 40 healthy 3-week-old specific pathogen-free male Sprague-Dawley rats were randomly and equally divided into four groups： control group （corn oil）, DEHP group （500 mg/kg, dissolved in corn oil）, CYP group （80 mg/kg, dissolved in corn oil）, and combined exposure group （exposed to 500 mg/kg DEHP and 80 mg/kg CYP, dissolved in corn oil）. Rats were treated by gavage administration once a day for 30 days. Twenty-four hours after the last exposure, the animals were sacrificed. The body weight and the wet weight of testis were determined, and the weight coefficient of testis was calculated. Radioimmunoassay was used to determine serum testosterone level. Uhrastrnctural-level histopathological changes of the testis were examined by transmission electron microscopy. The mRNA and protein expression of follicle stimulating hormone receptor （FSHR）, androgen binding protein （ABP）, inhibin beta-B （INHBB） and vimentin （VIM） were analyzed by real-time PCR and Western blot, respectively. Factorial design analysis of variance was used to compare differences between groups; interaction diagrams were used to determine the interaction between DEHP and CYP. Results Compared with those of the control group, the testis weights and testis coefficients of the DEHP, CYP, and combined exposure groups significantly decreased by 39.3-59.2% and 19.7-58.6%, respectively, and all exposure groups showed significant reductions in serum level of testosterone, ranging from 49.1% to 62.7% （P〈O.05 orP〈0.O1）. And all the exposure groups showed different levels of uhrastruetural damages in the testes. Compared with that in the control group, the mRNA expression of FSHR, ABP, INHBB, and VIM in the DEHP group was clown-regulated by 1.72, 2.64, 2.83 and 1.79 times, and their protein levels were significantly reduced by 65.2%, 53.7%, 70.1%, and 51.9% （P〈0.05 or P〈0.O1 ）. Significant decreases in mRNA expression of ABP （down 1.72 times） and INHBB （down 2.06 times） were observed in the CYP group, and their protein levels decreased by 38.3% and 49.7%, respectively （P〈0.05）. The combined exposure to both DEHP and CYP resulted in big decreases in the mRNA levels of FSHR （down 1.62 times）, ABP （down 2.00 times）, INHBB （down 2.35 times）, and VIM （down 1.54 times） and protein levels of FSHR （down 52.1＇7o）, INHBB （down 53.9%）, and VIM （down 58.8%） （P〈0.05）. Factorial design analysis of variance showed that the combination of two substances had an antagonistic effect on the expression of ABP and INHBB （P〈0.05）. Conclusion DEHP and CYP, alone or combined, can lead to gonadal dysgenesis in prepubertal male rats. Both of them can disrupt functional mRNA and protein expression in Sertoli cells to certain levels. The combination of DEHP and CYP shows antagonistic effects, and DEHP has a stronger reproductive toxicity than CYP.