Concentration: response analysis of high throughput data obtained in embryos cultured in vitro in presence of a binary mixture of two antifungal azoles (triadimefon and flusilazole)

The Benchmark Dose (BMD) method is the scientifically most advanced approach in risk assessment for estimating the dose (BMD) associated with a predefined response and to predict mixture effects. The BMD approach considers dose–response information derived from fitting mathematical models to the experimental data. In order to evaluate the whole series of endpoints and to model complex parameters (produced by scaled responses depending from the severity of the effects or obtained by gene expression analysis), the software package PROAST is actually the most skilful method. PROAST, in fact, has been developed for applications in concentration–response data from in vitro studies and to analyse high throughput data (gene expression as a function of dose, scores of effect). In the present work, postimplantation rat embryos were cultured in vitro in presence of triadimefon, flusilazole or of their binary mixtures. Experimental data on branchial abnormalities were scored on the basis of the severity of the effect. The expression of some genes (CYP26a1 and CYP26c1, considered as possible markers of effect) was evaluated by using real time RT-PCR. The outcomes were modelled using the PROAST software. The mixture predicted effects, derived under the dose-additivity hypothesis, were compared with the experimental results. The analysis of results shows that parallel dose–response is not rejected by the significance test. As far as gene expression is concerned, CYP26c1 seems to be the best candidate effect marker.