邻苯二甲酸二乙基己酯及代谢产物MEHP对幼鼠睾丸组织TGF-β1表达和端粒酶活性的影响

目的:观察邻苯二甲酸二乙基己酯(DEHP)及代谢产物邻苯二酸-单-2-乙基己酯(MEHP)对幼鼠睾丸组织转化生长因子-β1(TGF-β1)表达和端粒酶活性的影响,探讨DEHP和MEHP损害生精功能可能的机制。方法:生后2周龄的Wistar雄性幼鼠96只,随机分8组,每组12只。随机选择1组行生理盐水[0.9%NS0.2 ml/(kg.d),喂养3周]灌胃,作为正常对照组(NC组);再选1组行环磷酰胺[CTX 100 mg/(kg.d),喂养1周]灌胃,作为阳性对照组(PC组);余各组分别用DEHP、MEHP按低剂量[100 mg/(kg.d),喂养3周]、中剂量[200 mg/(kg.d),喂养2周]、高剂量[300 mg/(kg.d),喂养1周]灌胃制作动物模型。观察不同时期、不同剂量下睾丸组织精子形态变化,光镜下计数精子头部及畸形率;应用免疫组化SABC法及RT-PCR法检查睾丸组织TGF-β1的表达,并测定面密度;ELISA法检查睾丸组织中端粒酶活性。结果:①睾丸组织内精子形态变化:用药组精子数量减少,出现精子断头、无钩、双尾等畸形精子;在光镜下精子计数,与NC组比较,用药各组精子头部显著减少(P<0.05),畸形率增加(P<0.05),但高剂量短时间用药组与低剂量长时间用药组比较,差异无统计学意义(P>0.05)。②睾丸组织TGF-β1的表达变化:NC组低表达,DEHP及代谢产物MEHP染毒各组生精细胞内表达增多,PC组大量表达,阳性细胞呈黄褐色,主要分布于胞膜及胞质。NC组面密度为0.156 0±0.003 5、TGF-β1mRNA为1.51±0.20,PC组面密度为0.534 0±0.003 1、TGF-β1 mRNA为8.43±1.75;用药的DEHP组面密度均值为0.289 0±0.003 6、TGF-β1 mRNA为3.83±1.57,MEHP组面密度均值为0.284 0±0.003 1、TGF-β1 mRNA为3.51±1.41,用药各组与NC组、PC组比较差异有统计学意义(P<0.01),但高剂量短时间用药组与低剂量长时间用药组比较,差异无统计学意义(P>0.05);③睾丸组织中端粒酶活性:与NC组比较,用药各组睾丸组织中端粒酶活性下降(P<0.05),高剂量短时间用药组与低剂量长时间用药组比较,差异无统计学意义(P>0.05)。结论:DEHP及代谢产物MEHP对幼鼠生精功能有明显损害,其损害机制可能与DEHP及代谢产物MEHP诱导睾丸组织TGF-β1的表达水平升高、端粒酶活性降低有关。

Di-2-ethylhexyl phthalate and its metabolite single-ethylhexyl phthalate affect TGF-beta 1 expression and telomerase activity in the testis of young male rats

Objective: To investigate the influences of di-2-ethylhexyl phthalate(DEHP) and its metabolite single-ethylhexyl phthalate(MEHP) on the expression of transforming growth factor-beta 1(TGF-β1) and telomerase activity in young male Wistar rats.Methods: Ninety-six 2-week-old male Wistar rats were equally randomized into a normal control(NC) group,a positive control(PC) group,and six experimental groups.Those of the NC group were intragastrically administered 0.9% normal saline at a dose of 0.2 ml per kg per d for 3 weeks,those in the PC group cyclophosphamide(CTX) at 100 mg per kg per d for 1 week,and those of the experimental groups DEHP and MEHP,respectively,at a low dose(100 mg per kg per d) for 3 weeks,a moderate dose(200 mg per kg per d) for 2 weeks,and a high dose(300 mg per kg per d) for 1 week.Then we observed the morphological changes of the testicular sperm and counted the sperm heads and their abnormity rate at different doses and times.We detected the expression of TGF-β1 in the testis tissue using immunohistochemical SABC and RT-PCR,measured the area density,and determined telomerase activity by ELISA.Results: Compared with the NC group,the experimental groups showed an obvious reduction in the total sperm count and number of sperm heads(P<0.05) and a significant increase in the rate of teratosperm(P<0.05),such as decapitated,hookless,and double-tailed sperm.And there were no significant differences between the high-dose short-term and low-dose long-term medication groups(P>0.05).The expression of TGF-β1 was low in the NC group,high in the PC group,and obviously increased in the membrane and cytoplasm of spermatogenic cells of the experimental groups.The area density and TGF-β1 mRNA expression were 0.156 0±0.003 5 and 1.51±0.20 in the NC group,0.534 0±0.003 1 and 8.43±1.75 in the PC group,0.289 0±0.003 6 and 3.83±1.57 in the DEHP groups,and 0.284 0±0.003 1 and 3.51±1.41 in the MEHP groups.There were significant differences between the experimental and the other two groups(P<0.01),but not between the high-dose short-term and low-dose long-term medication groups(P>0.05).Telomerase activity was remarkably reduced in the experimental groups as compared with the NC group(P<0.05),but with no significant difference between the high-dose short-term and low-dose long-term medication groups(P>0.05).Conclusion: DEHP and its metabolite MEHP can evidently induce spermatogenic injury in young male rats,which may be associated with their induction of increased TGF-β1 expression and decreased telomerase activity in the rat testis.