ACROPOLIS. Overview on in vitro tests for craniofacial malformations. Giavini E., Menegola E., Di Renzo F.

Studies in vitro: postimplantation rat whole embryo culture E9.5 rat 48/60h Medium: rat serum triadimefon µM triadimenol µM flusilazole µM tebuconazole µM cyproconazole µM fluconazole µM triazole 5000 µM imazalil µM ketoconazole µM imidazole 1000 µM

In vitro tested azoles: In vivo tested azoles: Triadimefon, Ketoconazole, Cyproconazole Fluconazole, Itraconazole Craniofacial malformations ControlAzoles Control Azoles triadimefon µM triadimenol µM flusilazole µM cyproconazole µM fluconazole µM tebuconazole µM imazalil µM ketoconazole µM Fusion and reduction of the 1° and 2° branchial arches In vitro tested azoles

Normal Neural Crest cell migrationAbnormal neural crest cell igration Pathogenic pathway

Control Triadimefon

Azole inhibition

Undirect evidences that RA is implied in Azole-induced CM: 1)The craniofacial malformations induced by azoles are similar to those produced by excess of RA; 2)The co-exposure of rat embryos in vitro to subteratogenic concentrations of RA and fluconazole resulted in malformations; 3)The co-exposure to a teratogenic concentration of fluconazole and to citral (inhibitor of RA synthesis) resulted in a reduction of malformations; 4)The exposure to embryos to azole fungicides produced an increased expression of Cyp26. Conclusion: the azoles induce CM with the same pathway and the same mechanism of action.

The tested azole belong to a chemical family with: a)similar chemical structure; b) same mechanism of pesticide action; c)same target of toxic (teratogenic) effects; d)same pathogenic pathway; e)same mechanism of toxicological action (?) Cumulative assessment group for teratogenesis

Fig. 1. Percentage of branchial arch abnormalities (partial or total fusion between I and II branchial arches) and plurimalformed embryos in controls and in embryos exposed in vitro to tebuconazole or cyproconazole.

Fig. 2. Percentage of branchial arch abnormalities in controls and in embryos exposed in vitro to MIX2 and MIX3. Mix2: triadimefon + imazlil (NOEL concentrations) Mix3: triadimefon+imazalil+fluconazole (NOEL concentrations)

Future perspectives Next 6 months: To test in vitro some other azoles (e.g. propiconazole and prochloraz) In vitro study of teratogenic effects of mixtures: a) evaluation of Mix –dependence using a different number of azoles b) to verify the best dose approach (benchmark dose, fractions of NOELs etc.) c) identification of molecular biomarkers of embryotoxicity (Krox20, Tgfβ, Cyp26) Next years: Effects of mixtures of azoles administered to pregnant mice and rats a) fetal morphological abnormalities (in particular craniofacial); b) confirmation of validity of biomarkers in in vivo exposure.