Study On The Antiandrogenic Activity And Mechanism Of Six Pyrethroids

[Objective] Environmental antiandrogen,which belong to the environmtantal endocrine disrupters,can cause feminization and genital malformation of male animals,manifested in characteristics such as female-like anogenital distance,retained nipples,cleft phallus with hypospadias and reduced or absent sex accessory glands. It is clear that androgens are critical to sexual differentiation and reproduction in males,and the biological effects of them act through the AR undoubtedly.To date,most environmental antiandrogens are found to be capable of interaction with AR. Structure related pyrethroids including bifenthrin, permethrin, cypermethrin, beta-cypermethrin, cyfluthrin, beta-cyfluthrin are widely used in china and the potential for human exposure to this class of compounds is significant.Recently the antiandrogenic activity of permethrin has been reported.Herein we conducted in vivo and in vitro assays to investigate the Suspected antiandrogenic activity and mechanism of the six compounds.[Methods] Transcriptional activation assay was used to determine the AR antagonism effects of six pyrethroids in vitro utilizing the stably expressing androgen-responsive reporter gene cell line — MDA-kb2.MDA-kb2 cells stably transformed with the pMMTV.neo.luc reporter gene construct were maintained in L-15 media supplemented with 10% FBS,100U/ml penicillin, 100μg/ml streptomycin,and 0.25μg/ml amphotericin B at 37℃, without CO₂.This cell line was derived form the human breast-cancer cell MDA-MB-453,which contained endogenous androgen receptors. For experiments,MDA-kb2 cells were plated at 1 X104 cells per well in lOOul of medium in 96-well plates. When cells were attached,medium was removed and replaced with dosing mediumtthe six pyrethroids in the concentration range of 1.0 X 10"8mol/L — 1.0 X 10"5mol/L,or known antiandrogen-hydroxyflutamide with adding the potent,classical AR agonist,DHT at the same time.Dosing medium was prepared at the time of treatment by aliquoting 1 ul of stock solution into 1 ml mixed medium.Ethanol concentrations in media never exceeded 0.1%.Each plate also contained l.OnM DHT,or luM OHF as agonist and antagonist controls respectively.Cells were incubated overnight at 37^ without CO2.After 24h incubation,dosing media were then removed from each plate by inversion and gentle shaking,cells were washed once with phosphate-buffered saline at room temperature,and then were lysed using lysis buffer,at last luciferase activity was measured. Finally the fold inductions over vehicle control were calculated as standard luciferase activity value, which represents antiandrogenic potency.Because each cell in this stable cell line is derived from a single clone,responses are much less variable than those seen with transient transfection systems. Hershbergerassay was used to determine the antiandrogenic potential of the pyrethroids in vivo.SD male rats were surgically castrated at four weeks of age.At six weeks of age,they were randomly divided into several groups, each of which consisted of five rats. Each group of rats were administrated once daily for 7 days with testosterone propionate (lOOug , sc), excluding an additional group without TP, plus gavage dosage of pyrethroids as follows: bifenthrin at 1.5, 4.5, 13.5 mg/ (kg *d), permethrin at 3, 9, 27 mg/ (kg ? d), cypermethrin at 7> 21 , 63 mg/ (kg ? d), beta-cypermethrin at 2.5, 7.5, 22.5 mg/ (kg-d), cyfluthrinat 6, 18> 54mg/(kg ?d), beta-cyfluthrin at 4, 12, 36 mg/(kg ?d), flutamide at 50 mg/(kg *d), while hormone deficient control and vehicle control groups of rats were gavaged with same volumes of corn oil. After 7-day treatments, all rats were euthanized, and liver, spleen, kidney, adrenal, ventral prostate, dorsolateral prostate, seminal vesicle, cowper's glands, glans penis.preputial gland were excised and weighed respectively.[Results] In in vivo, compared with vehicle control, bifenthrin at dose of13.5 mg/ (kg 'dX cyfluthrin at dose of 18 and 54 mg/ (kg ?dX beta-cyfluthrin at dose of 12 and 36 mg/ (kg *d)> permethrin at dose of 45 and 135 mg/ (kg *d) caused significant decrease in some major androgen-responsive tissues;while in in vitro,bifenthrin could block DHT-induced AR activity in MDA-kb2 cells in a dose-dependent manner,at dose ranging form 1.0 X10"8 mol/L -1.0X10"5 mol/L without causing obvious cell toxicity. Cyfluthrin and beta-cyfluthrin decreased lnM DHT-induced luciferase activity in a dose-dependent manner at concentrations from 1.0 X10"8 mol/L to 1.0 X 10"6 mol/L, 1.0 X10"5 mol/L beta-cyfluthrin also significantly reduced DHT-mediated transcription compared to 1 .ODHT alone,but less dramatically than at 1.0 X 10*6 mol/L;unexpectedly,1.0 X 10'5 mol/L beta-cyfluthrin didn't show a significant effect of reduction. As reported,some compounds displayed mixed agonist and antagonist activity.The dual estrogenic/antiandrogenic activity may attribute to this phenomenon.[Conclusion] CyfluthrnK beta-cyfluthrin > permethrin > bifenthrin areenvironmental antiandrogen, and AR antagonism may contribute to the their antiandrogenic activity except permethrin,which mechanism of androgen antagonist remains to be study furthermore.The antiandrogenic activity may occur before significant general toxicity presents.