Azole pharmaceuticals induce germinal vesicle breakdown (GVBD) in preovulatory oocytes of zebrafish (Danio rerio): an in vitro study.

Abstract

Azoles, the antifungal pharmaceuticals are emerging as a new class of water contaminants with a potential to influence the endocrine physiology of surrounding aquatic fauna. In this study, we made an attempt to assess the relative efficacy of widely used azoles belonging to two subclasses, i.e., (i) triazoles (letrozole, fluconazole, itraconazole) and (ii) imidazoles (ketaconazole, ornidazole, clotrimazole), on the onset of germinal vesicle breakdown (GVBD) (an initial step in the final maturation of oocytes) in fully grown preovulatory oocytes of zebrafish (Danio rerio) using an in vitro model. Oocytes (> 650 μm) isolated manually from gravid ovaries were exposed to (i) 0.01 and/or 0.1, 1.0, 5.0, 10, 15, and 20 ng/ml and (ii) 1.0, 2.0, 3.0, 4.0, and 5.0 μg/ml of drugs. Zebrafish Ringer's solution (vehicle) and 0.01%ethyl alcohol (solvent) were used as negative controls. 17α, 20 β-Dihydroxy-4-pregnen-3-one (17α-DHP) and diethylstibestrol (DES), potent inducers of GVBD in fish, were used as positive controls. GVBD was scored hourly from 0-6 h. In negative controls, there were no indications of GVBD even at the 6th hour, while in 17α-DHP- and DES-exposed oocytes, GVBD was initiated from the 1st hour, reaching 80% and 76% respectively at the 6th hour. Among azoles, letrozole induced GVBD in 73-85%, fluconazole (30-33%), itraconazole (23-33%), ketaconazole (46-53%), ornidazole (36-40%), and clotrimazole (30-33%) of oocytes. These results suggest that azole pharmaceuticals induce GVBD in fish oocytes that may be attributed to their variable degree of cytochrome P450 enzyme inhibitor activity.