Study of the mixture effects of two compounds with different mode of action (fluconazole and ethanol) by using Ciona intestinalis as a new alternative animal model

The aim of the present work is to evaluate the effects of the co-exposure to ethanol (Eth) and to the clinically used fungicide fluconazole (FLUCO) by using a new alternative model for teratological screening: Ciona intestinalis. The simple development of ascidians (Chordata, Tunicata) and their key phylogenetic position within the sister group of vertebrates, suggests the ascidian model as a potential good alternative experimental system. C. intestinalis embryos were exposed to FLUCO (31.5-62.5-125-250-500 µM), to Eth at its not effect level (0.1%) or to the mixture of FLUCO and Eth from 2-cell to the larval stage. At the end of the culture period, larvae were morphologically examined. Eth alone was unable to affect embryo development. Larvae exposed to FLUCO showed a typical phenotype characterized by malformations at the trunk region comparable to those elicited by retinoic acid (RA). The trunk appeared round in shape with flat palps, the sensory vesicle cavity was absent or reduced and, therefore, both pigmented cells, otolith and ocellus, were dislocated in the dorsal portion. The effects were FLUCO concentration-related. Interestingly, a significant increase of larvae with severe malformations was observed in groups co-exposed to FLUCO and Eth: almost the 100% of larvae showed a severely affected phenotype characterized by absence of sensory vesicle cavity, by absence of pigment in the sensory organs and presence of a short, curled and thick tail. The anterior end was round in shape, the palps were not elongated, and larvae failed the hatching. The obtained data point the attention on the teratogenic risk of co-exposure to FLUCO and Eth. These results are comparable to those previously obtained in postimplantation rat embryo cultured in vitro. The evidence that different subphyla (tunicates and vertebrates) are susceptible to azole fungicides and that the observed effects are quite similar, suggests the intriguing hypothesis that these molecules alter the expression of ancestral conservative genes, starter for a cascades of events, which model the whole embryonic body plan. Finally, ascidian embryo seems to be a good experimental system for a comparative screening of the teratogenic potential of azole fungicide mixtures, pointing the attention on possible environmental impact of azole fungicides.