邻苯二甲酸二丁酯诱导氧化应激及抑制CYP17a1干扰睾酮合成

Testicular oxidative stress and downregulation of CYP17a1 indused by di(n-butyl) phthalate inhibit synthesis of testosterone

目的探讨邻苯二甲酸二丁酯(DBP)导致睾丸氧化损伤与睾酮合成途径的关系及可能的机制。方法 24只健康4周龄雄性Wistar大鼠随机分为对照组(玉米油)及DBP低、中、高剂量组(80、200和500 mg/kg),每组6只,经灌胃染毒每日1次、连续染毒4周。染毒结束后称量动物体重及睾丸和附睾的重量,用生化方法检测睾丸匀浆中丙二醛(MDA)和活性氧自由基(ROS)的含量,抗氧化酶超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶-1(GPx-1)的活性变化,类固醇合成酶3β-HSD1、17β-HSD3的活性,用放射免疫法测定外周血中睾酮、促黄体生成素(LH)、卵泡刺激素(FSH)和睾丸内的睾酮(T)、雄烯二酮(ASD)的水平,用real timeq PCR方法检测St AR、P450scc、3β-HSD1、17β-HSD3、CYP17a1 mRNA水平的表达变化。结果与对照组相比,高剂量组体重、睾丸重量明显减少(P<0.05);循环血LH、FSH增高(P<0.05),循环血及睾丸内睾酮、睾丸内ASD均降低(P<0.05);MDA和ROS的含量明显升高(P<0.05),SOD、CAT、GPx-1的活性及3β-HSD1的酶活性均明显降低(P<0.05);St AR、P450scc、3β-HSD1、CYP17a1 mRNA降低,17β-HSD3 mRNA升高(P<0.05)。中剂量组循环血LH、FSH增高(P<0.05),睾丸内T和ASD降低(P<0.05);ROS升高(P<0.05),GPx-1的活性降低(P<0.05);3β-HSD1酶活性减少(P<0.05);St AR、P450scc、CYP17a1 mRNA降低(P<0.05)。低剂量组所有指标未见有统计学意义的改变。结论 DBP暴露干扰了大鼠睾丸氧化/抗氧化平衡,降低了GPx-1活性,抑制CYP17a1基因的表达,降低睾丸ASD水平,最终抑制间质细胞内睾酮合成。CYP17a1降低是DBP干扰睾酮合成的毒性作用机制之一。

Objective To explore the relationship and possible mechanisms between testicular oxidative injury caused by DBP and the testosterone synthesis pathway. Methods Twenty-four male Wistar male rats （4-wk-old） were randomly divided into 4 groups , three doses of DBP （80, 200 and 500 mg/kg） groups and a vehicle （corn oil） control group, 6 animals each. The rats were respectively administered by garage once a day for four weeks. They were sacrificed after 4 weeks treatment and the body weights, testis, epididymis were weighed, respectively. The oxidation of MDA and ROS, the activity changes of antioxidases SOD, CAT and GPx-1, as well as the activities of steroid synthetases 3β-HSD1, 17β-HSD3 in the testis homogenate were measured by biochemical methods. The levels of testosterone, LH , FSH in peripheral blood and testosterone, ASD in testis were measured by radioimmunoassay. The intensities of expresses of STAR, P450scc, 3β-HSD1,17β-HSD3 and CYP17a1 mRNA were detected by real-time qPCR. Results In 500 mg/kg dose group, the body weights and weigths of testis were decreased obviously （P 〈 0. 05 ）. The concentration of serum LH and FSH was increased, the consentration of serum T, testicular T and testicular ASD was decreased （P 〈 0.05）. The oxidation of MDA and ROS was increased distinctly and the activities of SOD, CAT, GPx- 1 and 3β-HSD1 were reduced （P 〈 0. 05）. STAR, P450scc, 3β-HSD1 and CYP17a1 mRNA were decreased, 17β-HSD3 mRNA was increased （P 〈 0.05）. In 200mg/kg dose groups, LH , FSH level in peripheral blood were increased and ASD level in testis was decreased （ P 〈 0. 05 ）. The oxidation of ROS was increased and activity of GPx-1 and 3β- HSD1 were decreased （P 〈0.05）. STAR, P450scc and CYP17a1 mRNA were decreased （P 〈 0.05）. There were no changes in 80mg/kg group. Conclusion DBP exposure disturbed the balances of oxidation/antioxidation, then result in the decline of GPx-1 activity, CYP17a1 mRNA and ASD level which caused the decrease of testosterone synthesis in ledyig cell. It is speculated that the decrease of CYP17a1 may be one of the mechanisms of toxic effects of DBP. Key words： di（n-butyl） phthalate, leydig cells, oxidative damage, CYP17a1, steroidogenesis