Effect of the co-exposure of ethanol and fluconazole on in vitro post-implantation rat embryo development

The specific teratogenic effect of some azole fungicides, including fluconazole (FLUCO, clinically used), has been investigated in the past using postimplantation rat whole embryo culture. Embryos exposed to azoles develop abnormal branchial structures related to the abnormal hindbrain segmentation and abnormal neural crest cell (NCC) migration and compaction. The postulated mechanism of action is the inhibition of CYP26 enzymes (involved in retinoic acid (RA) degradation), with the consequent increase in RA local content. The aim of this work is to evaluate the influence of sub-teratogenic concentration of Ethanol (Eth, suspected to imbalance RA production in embryos) on FLUCO-related teratogenic effects. Rat embryos were exposed in vitro from the late gastrula/early neurula stage till the phylotytic stage to Eth alone (1 μL/mL culture medium), to FLUCO (62.5–125–250–500 μM), or co-exposed to FLUCO and Eth. At the end of the culture period, embryos were morphologically examined and processed for gene expression analysis in order to evaluate genes specifically involved in RA metabolism (ADH7, CRBP, CRABP, CYP26a1, CYP26b1, CYP26c1). Eth itself was unable to affect embryo development. Groups exposed to FLUCO 125–250–500 μM showed specific abnormalities at the level of the branchial apparatus, comparable to those previously documented and related to a probable RA increase. The effects were FLUCO concentration-related. FLUCO 62.5 was the no effect level (NOAEL). However, when embryos were co-exposed to Eth and FLUCO 62.5 μM abnormalities were observed. Also, a significant enhancement of teratogenic effects were observed in groups co-exposed to FLUCO (125–250–500 μM) and Eth. Gene expression revealed that both Eth and FLUCO 62.5 μM altered the gene expression in a manner consistent with a RA increase (up-regulation of the RA inducible CYP26a1, down-regulation of the RA not inducible CYP26c1). The co-exposure to FLUCO and Eth worsened the gene expression imbalance. The obtained data show that the co-exposure to Eth at sub-teratogenic level is able to enhance the FLUCO teratogenicity. The gene expression analysis suggests that the imbalance of RA production could be the basis of the observed combined effects of the two molecules.