Novel mechanism of endocrine disruption by fungicides through binding to the membrane androgen receptor, ZIP9 (SLC39A9), and antagonizing rapid testosterone induction of the intrinsic apoptotic pathway

A variety of pesticides including vinclozolin, its metabolite M2 (3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide), and prochloraz have been shown to exert antiandrogenic effects in animal models by competing with androgen binding to nuclear androgen receptors (nAR) and decreasing transcription of androgen-responsive genes. However, it is not known whether these pesticide antiandrogens also interfere with rapid (often described as nongenomic, nonclassical) androgen actions mediated by membrane androgen receptors (mARs). We recently discovered that ZIP9, a member of the zinc transporter ZIP (SLC39A) family, is a specific, high-affinity mAR that mediates rapid testosterone-dependent signaling, zinc influx, and apoptosis in breast and prostate cancer cell lines. Possible disruption by prochloraz, vinclozolin, and M2 of androgen actions through this mAR was investigated in vitro in PC-3 prostate cancer cells (nAR-) over expressing human ZIP9 (PC3-ZIP9 cells). Single point competitive binding assays showed 1 mu M and 10 mu M concentrations of all three pesticides displaced specific [H-3]-testosterone binding to PC3-ZIP9 cell membranes with binding affinities < 10% that of testosterone. The pesticides also exerted antiandrogen actions through ZIP9. Co-treatments with 100 nM prochloraz, vinclozolin and M2 blocked or attenuated the 20 nM testosterone-induced increases in apoptosis, intracellular free zinc levels, and expression of the proapoptotic gene, Bax. Prochloraz also attenuated testosterone activation of MAPkinase. The finding that prochloraz, vinclozolin and M2 are effective competitors of [H-3]-testosterone binding to ZIP9 and block testosterone actions mediated through ZIP9 in vitro at nanomolar concentrations suggests that androgen functions mediated by ZIP9 are also susceptible to disruption by pesticide antiandrogens with potential adverse effects on human health.