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
cyproconazole 15.625-250µM
fluconazole 62.5-500 µM
triazole 5000 µM
Medium: rat serum
E9.5 rat
imazalil 5-100 µM
ketoconazole 5-100 µM
imidazole 1000 µM
48/60h
In vitro tested azoles
In vivo tested azoles:
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
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
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)
b)
c)
d)
e)
similar chemical structure;
same mechanism of pesticide action;
same target of toxic (teratogenic) effects;
same pathogenic pathway;
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.