DEVELOPMENT OF AN INTEGRATED SYSTEM BIOLOGY MODEL FOR PREDICTING MIXTURES OF CHEMICALS ACTING ON THE SAME PATHWAY

Exposure of the embryo to environmental chemicals (pesticides, air and water pollutants) can result in congenital malformations or developmental defects such as oro-facial cleftings. Unfortunately, the human embryo is not usually exposed to a single substance, but to many substances simultaneously. Despite the efforts in elucidating mechanism of action (MoA) of substances that perturb the normal embryonic development, only a small part of involved pathways have been understood to date (Giavini and Menegola, 2004). This is why, evaluating the toxicity of mixtures of multiple chemicals is one of the major objectives of today\u2019s toxicology despite the effect of exposure to a mixture is still difficult to understand. To arrive in the future to the creation of a realistic overall picture of human exposure to mixtures, the development of integrated approaches between in vitro and in silico techniques and computational systems biology, able to predict the effects of mixtures starting from the concentrations of their individual components, will be essential. Recent studies suggest that the similarity of molecular initiating events (MIEs) is not an essential requirement to induce additive effects, because mixtures composed of chemicals with different MIE can exhibit mixture effects too, probably acting on the same biological pathway and contributing to the same adverse outcome (EFSA, 2013). This is in recognition of emerging evidence that biological effects can be similar, although the molecular details of toxicological mechanisms may profoundly differ in many respects (Kortenkamp, 2007). Considering the previously reported data, this could be the case of embryonic co-exposure to fluconazole (FLUCO) and ethanol (Eth). Both, in fact, lead to the same adverse outcome (AO), craniofacial malformations, both after in utero and in vitro exposure. The two molecules are able to induce cranio-facial defects (in embryos visible as cranio-facial abnormalities), probably acting on the same Adverse Outcome Pathway (AOP) altering, with different MIEs, the biological event cascade regulated by the morphogen retinoic acid (RA). The specific aim of this PhD project was to investigate this hypothesis through the development of an in silico model, useful to simulate and predict the effects on embryo development after co-exposure to substances with independent MoAs but acting on the same biological pathway and potentially contributing to the same adverse outcome (cranio-facial malformations). The in silico model was based on experimental data and validated by in vitro experiments. For this purpose, the project was divided into three parts. In the first part, we evaluated the effects of the molecules on post-implantation rat embryos cultures, using the in vitro technique WEC (Whole Embryo Culture). Embryos were cultured in presence of increasing concentration of RA (0.025-0.0375-0.05-0.125-0.25 \ub5M), to increasing concentrations of Eth (17-42.5-85-127 mM), to increasing concentrations of FLUCO (62.5-125-250-500 \ub5M). Specific and concentration related abnormalities at the level of the branchial arches (reductions or fusions) were observed after exposure to single Eth or FLUCO and were comparable to those elicited by RA. These results suggest a common AO for Eth, FLUCO and RA. Embryos were then co-exposed to binary mixtures of FLUCO and Eth. To better characterize the contribution of each component to the observed effects, the \u201cfixed + moving\u201d approach was applied: embryos were exposed to the no effect concentration (NOAEL) of one chemical (\u201cfixed\u201d) and increasing concentrations of the other chemical (\u201cmoving\u201d). A significant enhancement of teratogenic effects was observed after co-exposure to FLUCO and Eth in comparison to the single exposure. The mixture between the two NOAELs was effective too, inducing almost 40% of branchial arch abnormalities. These data suggest the presence of a cumulative effect in mixture, probably due to the capability of both molecules to perturb RA endogenous concentrations in specific tissues (the precursors of cranio-facial skeletal tissues, originated in the embryonic hindbrain). This theory could be explained considering the ability of Eth to induce alcohol-dehydrogenases (including ADH7, the embryonic enzyme involved in RA synthesis) and the inhibitory effects of FLUCO on cytochromes p450 (including CYP26, involved in embryonic RA degradation). In the second part of the project, we evaluated these hypothetical mechanisms inducing the Eth-FLUCO mixture effects through the development of an in silico tool, able to simulate both the formation of the physiological RA gradient in the rat embryo hindbrain and its perturbation after exposure to FLUCO, Eth and to their binary mixtures. The obtained system biology model, developed using an integrated approach combining mathematical modelling, molecular docking and in vitro experiments, seems to be reasonably predictive for the mixture\u2019s effects, confirming the accuracy of the hypothesized pathogenic pathway. Experimental data and model predictions, in fact, showed a promising agreement. The model, in spite of its limitations, could have many potential mechanistic or predictive applications for the study of risk assessment. However, since the model is based on experimental data obtained in mammals, the last part of the project was aimed to evaluate alternative animal models. In the third part of the project, the evaluation of the effects after co-exposure to FLUCO and Eth were performed using the ascidian Ciona intestinalis embryo model as a new alternative teratological screening test (AET, Ascidian Embryo Teratogenicity assay). An ascidian species was selected because Ascidiacea represent the sister group of Vertebrates. For this purpose, C. intestinalis embryos were exposed to Eth alone (1.7-8.5-17-42.5-85 mM) to FLUCO alone (7.8-15.75-31.5-250 \ub5M), or co-exposed to binary mixtures of FLUCO and Eth until the larval stage, applying the fix + moving protocol. Specific and concentration related abnormalities at the level of the anterior structures were elicited by Eth or FLUCO, and were comparable to those described in literature after RA exposure. A significant enhancement of the general teratogenicity was observed after co-exposure to FLUCO and Eth in comparison to the single exposure, suggesting the presence of a mixture effect induced by FLUCO and Eth also in this model. These results, similar to those observed in the WEC model, encourage the use of AET as a complementary alternative method for embryotoxicity studies on mixtures. The possibility to translate data obtained in this model in our in silico model is still to evaluate. In conclusion, our data suggest that: 1. the integrated use of data from in vitro and in silico approaches used in this study support the hypothesis that embryonic exposure to FLUCO and Eth can lead to the same AO (craniofacial abnormalities) acting with different MIEs but both converging on the same AOP by altering the RA production (Eth) and the RA catabolism (FLUCO); 2. the hypothesis that substances with different MoAs but acting on the same pathway could produce an additive effect also at concentrations considered not effective is supported; 3. the obtained results highlight the potential additive effect that could occur after exposure to azoles and ethanol, suggesting a precautionary position in alcohol consumption during azoles exposure in pregnancy. The overall view of the obtained results support the need of a cumulative risk assessment not only for chemicals grouped on the base of similarities in chemical structure or derived from mechanistic considerations but also for chemicals differently acting on the same biological pathway