Study of the mixture effects of fluconazole and ethanol by using Ciona intestinalis as a new alternative teratogenical model

The aim of the present work is to evaluate the effects of the co-exposure to sub-teratogenic levels of ethanol (Eth) and to the clinically used antimycotic 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 for teratological purposes. 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 colture 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 effects were FLUCO concentration-related. Interestingly, a significant increase of larvae with severe malformations was observed in groups co-exposed to FLUCO and Eth: the 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 tail. The anterior end was round in shape, the palps were not elongated, and larvae failed the hatching. The obtained data point the attention to the teratogenic risk of co-exposure to FLUCO and Eth. The results are comparable to those previously obtained in postimplantation rat embryo cultured in vitro and could be related to RA increase. Considering that FLUCO and Eth do not share the same mode of action (MOA), our data support the need of a cumulative risk assessment not only for chemicals grouped on the base of similarities in chemical structure but also for chemicals differently acting on the same biological pathway. The evidence that different subphyla (Tunicates and Vertebrates) are susceptible to azole fungicides and that the observed effects are quite similar, suggests the hypothesis that these molecules alter the expression of ancestral conservative genes, starter of 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 to a possible environmental impact of azole fungicides.