Conazole fungicides are widely used in agriculture despite their suspected endocrine disrupting properties. In this study, the potential (anti-)androgenic effects of ten conazoles were assessed and mutually compared with existing data. Effects of cyproconazole (CYPRO), fluconazole (FLUC), flusilazole (FLUS), hexaconazole (HEXA), myconazole (MYC), penconazole (PEN), prochloraz (PRO), tebuconazole (TEBU), triadimefon (TRIA), and triticonazole (TRIT) were examined using murine Leydig (MA-10) cells and human T47D-ARE cells stably transfected with an androgen responsive element and a firefly luciferase reporter gene. Six conazoles caused a decrease in basal testosterone (T) secretion by MA-10 cells varying from 61% up to 12% compared to vehicle-treated control. T secretion was concentration-dependently inhibited after exposure of MA-10 cells to several concentrations of FLUS (IC50 = 12.4 μM) or TEBU (IC50 = 2.4 μM) in combination with LH. The expression of steroidogenic and cholesterol biosynthesis genes was not changed by conazole exposure. Also, there were no changes in reactive oxygen species (ROS) formation that could explain the altered T secretion after exposure to conazoles. Nine conazoles decreased T-induced AR activation (IC50s ranging from 10.7 to 71.5 μM) and effect potencies (REPs) were calculated relative to the known AR antagonist flutamide (FLUT). FLUC had no effect on AR activation by T. FLUS was the most potent (REP = 3.61) and MYC the least potent (REP = 0.03) AR antagonist. All other conazoles had a comparable REP from 0.12 to 0.38. Our results show distinct in vitro anti-androgenic effects of several conazole fungicides arising from two mechanisms: inhibition of T secretion and AR antagonism, suggesting potential testicular toxic effects. These effects warrant further mechanistic investigation and clearly show the need for accurate exposure data in order to perform proper (human) risk assessment of this class of compounds.
Keywords: 17β-HSD3, 17β-hydroxysteroid dehydrogenase type 3; 3β-HSD1, 3β-hydroxysteroid dehydrogenase type 1; AR, androgen receptor; Androgen receptor (AR); BMR, benchmark response; CHO cells, Chinese hamster ovary cells; CYP19, cytochrome P450 enzyme 19 (aromatase); CYP51, cytochrome P450 enzyme 51/lanosterol 14α-demethylase; CYPRO, cyproconazole; Conazole fungicides; Cyp11A1, cytochrome P450 enzyme 11A; Cyp17, cytochrome P450 enzyme 17; Cyproconazole (PubChem CID: 86132); DMEM, Dulbecco's Modified Eagle Medium; EC50, half maximal effective concentration; EDCs, endocrine disrupting chemicals; Endocrine disrupting chemicals (EDCs); FLUC, fluconazole; FLUS, flusilazole; FLUT, flutamide; FP, forward primer; FSH(R), follicle-stimulating hormone (receptor); Fluconazole (PubChem CID: 3365); Flusilazole (PubChem CID: 73675); H295R, human adrenocortical carcinoma cells; HEXA, hexaconazole; HMG-CoA red, HMG-CoA reductase; HSD(s), hydroxysteroid dehydrogenase(s); Hexaconazole (PubChem CID: 66461); IC50, half maximal inhibitory concentration; LH(R), luteinizing hormone (receptor); MA-10 Leydig cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MYC, myclobutanil; Myclobutanil (PubChem CID: 6336); NCBI, National Center for Biotechnology Information; PBS, phosphate-buffered saline; PEN, penconazole; PRO, prochloraz; Penconazole (PubChem CID: 91693); Por, cytochrome P450 oxidoreductase; Prochloraz (PubChem CID: 73665); REP, relative effect potency; RIA, radioimmunoassay; ROS, reactive oxygen species; RP, reverse primer; RT-qPCR, real time quantitative polymerase chain reaction; Spermatogenesis; StAR, steroidogenic acute regulatory protein; T, testosterone; TEBU, tebuconazole; TRIA, triadimefon; TRIT, triticonazole; Tebuconazole (PubChem CID: 86102); Testosterone (T); Triadimefon (PubChem CID: 39385); Triticonazole (PubChem CID: 6537961); cAMP, 8-bromoadenosine 3′,5′-cyclic monophosphate.