110 likes | 261 Views
ACROPOLIS. Overview on in vitro tests for craniofacial malformations. Giavini E., Menegola E., Di Renzo F. Studies in vitro: postimplantation rat whole embryo culture. triadimefon 12.5-250 µM triadimenol 6.25-125 µM flusilazole 3.125-25 µM tebuconazole 31.25-500µM
E N D
ACROPOLIS. Overview on in vitro tests for craniofacial malformations. Giavini E., Menegola E., Di Renzo F.
Studies in vitro: postimplantation rat whole embryo culture triadimefon12.5-250 µM triadimenol6.25-125 µM flusilazole 3.125-25 µM tebuconazole 31.25-500µM cyproconazole 15.625-250µM fluconazole62.5-500 µM triazole 5000 µM Medium: rat serum imazalil5-100 µM ketoconazole5-100 µM imidazole 1000 µM E9.5 rat 48/60h
In vitro tested azoles: In vitro testedazoles triadimefon 25-250 µM triadimenol 12.5-125 µM flusilazole 6.25-25 µM cyproconazole 15.625-250µM fluconazole 125-500 µM tebuconazole 31.25-500µM imazalil 5-10-50-100 µM ketoconazole 5-10-50-100 µM Fusion and reduction of the 1° and 2° branchial arches In vivo tested azoles: Triadimefon, Ketoconazole, Cyproconazole Fluconazole, Itraconazole Craniofacial malformations Control Azoles Control Azoles
Pathogenic pathway Normal Neural Crest cell migration Abnormal neural crest cell igration
Control Triadimefon
Undirectevidencesthat RA isimplied in Azole-inducedCM: • The craniofacialmalformationsinducedbyazoles are similartothoseproducedbyexcessof RA; • The co-exposureofratembryosin vitro tosubteratogenicconcentrationsof RA and fluconazoleresulted in malformations; • The co-exposureto a teratogenicconcentrationoffluconazole and tocitral (inhibitorof RA synthesis) resulted in a reductionofmalformations; • The exposuretoembryostoazolefungicidesproducedanincreasedexpressionof Cyp26. Conclusion: the azoles induce CM with the same pathway and the same mechanism of action.
The testedazolebelongto a chemical family with: • similarchemicalstructure; • samemechanismofpesticideaction; • same target oftoxic (teratogenic) effects; • samepathogenicpathway; • samemechanismoftoxicologicalaction (?) • Cumulative assessmentgroupforteratogenesis
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 otherazoles (e.g. propiconazole and prochloraz) • In vitro studyofteratogeniceffectsofmixtures: • a) evaluationof Mix –dependenceusing a differentnumberofazoles • b) toverify the best dose approach (benchmark dose, fractionsofNOELs etc.) • c) identificationofmolecularbiomarkersofembryotoxicity (Krox20, Tgfβ, Cyp26) • Nextyears: • Effectsofmixturesofazolesadministeredtopregnantmice and rats • a) fetalmorphologicalabnormalities (in particularcraniofacial); • b) confirmationofvalidityofbiomarkers in in vivo exposure.