Schrader T J, Cooke G M
Toxicology Research Division, Food Directorate, Health Canada.
Toxicol Sci. 2000 Feb;53(2):278-88. doi: 10.1093/toxsci/53.2.278.
In order to produce a reporter gene assay for androgenic chemicals, a constitutive expression vector coding for the human androgen receptor and a reporter construct containing the firefly luciferase coding sequence under transcriptional control of the androgen responsive MMTV promoter were cotransfected into the androgen-insensitive human PC-3 prostate carcinoma cell line and stable transfectants selected. One colony of transfectants, PC-3 LUCAR+, was characterized further. 5alpha-Dihydrotestosterone (DHT) enhanced luciferase activity in a linear fashion for up to 3 days of culture. The Kd for DHT activation was within the range of 25.0-60.0 pM (r2 values >0.95). Flutamide competitively inhibited DHT activation (mean Ki value of 0.89 microM). Progesterone, estradiol, dexamethasone, and hydrocortisone were weak agonists (100-fold less effective than DHT) and diethylstilbestrol was without effect. The effects of organochlorine food contaminants (0, 0.1, 1.0, and 10.0 microM) on luciferase activity in PC-3 LUCAR+ cells were determined after exposure to the chemical for 18 h in the presence and absence of DHT (50 pM). 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p'-DDE) induced luciferase activity in the absence of DHT (100 microM p,p'-DDE equivalent to 50 pM DHT), but in the presence of DHT (50 pM), p,p'-DDE acted antagonistically. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, kepone, butylated hydroxyanisole, and butylated hydroxytoluene all partially inhibited activation by DHT (50 pM) but alone had little or no effect. Toxaphene at 10 microM induced luciferase activity in the absence of DHT but decreased cell viability. Alpha- and delta-Hexachlorocyclohexanes (HCH) at 10 microM antagonized the DHT effect, but beta-HCH and gamma-HCH mirex, photomirex, oxychlordane, cis- and trans-nonachlor were without effect. Thus, of the chemicals tested, some interact with the human androgen receptor in vitro as agonists, others as antagonists, and some as partial agonists/antagonists.
为了建立一种针对雄激素类化学物质的报告基因检测方法，将编码人雄激素受体的组成型表达载体和一个报告构建体共转染到雄激素不敏感的人PC-3前列腺癌细胞系中，该报告构建体包含在雄激素反应性MMTV启动子转录控制下的萤火虫荧光素酶编码序列，并筛选出稳定转染子。对其中一个转染子菌落PC-3 LUCAR+进行了进一步表征。在培养长达3天的时间里，5α-二氢睾酮（DHT）以线性方式增强荧光素酶活性。DHT激活的解离常数（Kd）在25.0 - 60.0 pM范围内（r2值>0.95）。氟他胺竞争性抑制DHT激活（平均Ki值为0.89 microM）。孕酮、雌二醇、地塞米松和氢化可的松是弱激动剂（效力比DHT低100倍），己烯雌酚则无作用。在存在和不存在DHT（50 pM）的情况下，将有机氯食品污染物（0、0.1、1.0和10.0 microM）作用于PC-3 LUCAR+细胞18小时后，测定其对荧光素酶活性的影响。1,1-二氯-2,2-双（对氯苯基）乙烯（p,p'-DDE）在不存在DHT的情况下诱导荧光素酶活性（100 microM p,p'-DDE相当于50 pM DHT），但在存在DHT（50 pM）时，p,p'-DDE起拮抗作用。2,3,7,8-四氯二苯并-p-二恶英、开蓬、丁基羟基茴香醚和丁基羟基甲苯都部分抑制DHT（50 pM）的激活作用，但单独使用时作用很小或无作用。10 microM的毒杀芬在不存在DHT的情况下诱导荧光素酶活性，但会降低细胞活力。10 microM的α-和δ-六氯环己烷（HCH）拮抗DHT的作用，但β-HCH、γ-HCH、灭蚁灵、光灭蚁灵、氧氯丹、顺式和反式九氯则无作用。因此，在所测试的化学物质中，一些在体外与人雄激素受体相互作用时表现为激动剂，一些为拮抗剂，还有一些为部分激动剂/拮抗剂。
