Kizu Ryoichi, Okamura Kazumasa, Toriba Akira, Mizokami Atsushi, Burnstein Kerry L, Klinge Carolyn M, Hayakawa Kazuichi
Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-0934, Japan.
Toxicol Sci. 2003 Dec;76(2):299-309. doi: 10.1093/toxsci/kfg230. Epub 2003 Sep 11.
We collected diesel exhaust particles (DEPs) emitted from three diesel-engine vehicles--a car, a bus, and a truck--in daily use, and prepared DEP extracts (DEPEs), designated as EC, EB, or ET, respectively. The androgenic and antiandrogenic effects of the DEPE samples were examined by a luciferase reporter assay in human prostate carcinoma PC3/AR cells transiently transfected with a prostate specific antigen gene promoter-driven luciferase expression vector pGLPSA5.8. PC3/AR is a subline of human prostate carcinoma PC3 transformed to stably express wild-type human androgen receptor (AR). While DEPE samples did not exhibit any androgenic effect, they exerted antiandrogenic effect, inhibiting dihydrotestosterone (10 pM) -induced luciferase activity by 24 to 52% at an extract concentration of 10 microg/ml. The antiandrogenic effect was greater in the following order: ET > EB > EC. Co-treatment of PC3/AR cells with SKF-525A, a nonselective inhibitor of cytochrome P450 (CYP) enzymes, enhanced the antiandrogenic effect, indicating that the antiandrogenic effect is caused by intact species of DEPE constituents. The antiandrogenic effect of DEPE samples was reversed by alpha-naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist. The antiandrogenic activity of a DEPE sample correlated with its AhR agonist activity assayed in PC3/AR cells transiently transfected with CYP1A1 gene promoter-driven luciferase expression vector pLUC1A1. Equimolar mixtures of ten polycyclic aromatic hydrocarbons (PAHs) having four or more rings, structures found in the DEPEs, showed significant antiandrogenic effects and AhR agonist activity at concentrations equivalent to those found in DEPE samples. Further, DEPE samples elicited only antiandrogenic effects in recombinant yeast cells, which express beta-galactosidase in response to androgen. A competitive AR binding assay showed that AR-binding constituents exist in DEPE samples, indicating that greater part of AR-binding constituents in DEPEs are AR antagonists. All these findings show that DEPE samples exhibit significant antiandrogenic effect in cell-based transcription assay and that this effect is due in part to the constituents with AhR agonist activity including PAHs and to the constituents with AR antagonist activity.
我们收集了三辆日常使用的柴油发动机车辆（一辆轿车、一辆公交车和一辆卡车）排放的柴油尾气颗粒（DEP），并制备了DEP提取物（DEPE），分别命名为EC、EB或ET。通过荧光素酶报告基因检测法，在瞬时转染了前列腺特异性抗原基因启动子驱动的荧光素酶表达载体pGLPSA5.8的人前列腺癌PC3/AR细胞中，检测DEPE样品的雄激素作用和抗雄激素作用。PC3/AR是经转化后稳定表达野生型人雄激素受体（AR）的人前列腺癌PC3的一个亚系。虽然DEPE样品未表现出任何雄激素作用，但它们发挥了抗雄激素作用，在提取物浓度为10微克/毫升时，可将二氢睾酮（10皮摩尔）诱导的荧光素酶活性抑制24%至52%。抗雄激素作用按以下顺序增强：ET > EB > EC。用细胞色素P450（CYP）酶的非选择性抑制剂SKF-525A对PC3/AR细胞进行共处理，增强了抗雄激素作用，表明抗雄激素作用是由DEPE成分的完整种类引起的。DEPE样品的抗雄激素作用被芳基烃受体（AhR）拮抗剂α-萘黄酮逆转。在瞬时转染了CYP1A1基因启动子驱动的荧光素酶表达载体pLUC1A1的PC3/AR细胞中，DEPE样品的抗雄激素活性与其AhR激动剂活性相关。十种具有四个或更多环的多环芳烃（PAH）的等摩尔混合物（这些结构存在于DEPE中），在与DEPE样品中发现的浓度相当的浓度下，表现出显著的抗雄激素作用和AhR激动剂活性。此外，DEPE样品在重组酵母细胞中仅引起抗雄激素作用，这些细胞在雄激素作用下表达β-半乳糖苷酶。竞争性AR结合试验表明，DEPE样品中存在AR结合成分，这表明DEPE中大部分AR结合成分是AR拮抗剂。所有这些发现表明，DEPE样品在基于细胞的转录试验中表现出显著的抗雄激素作用，并且这种作用部分归因于具有AhR激动剂活性的成分（包括PAH）和具有AR拮抗剂活性的成分。
