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 12.5-250 µMtriadimenol 6.25-125 µMflusilazole 3.125-25 µMtebuconazole 31.25-500µMcyproconazole 15.625-250µMfluconazole 62.5-500 µMtriazole 5000 µM

imazalil 5-100 µMketoconazole 5-100 µMimidazole 1000 µM

In vitro tested azoles:

In vivo tested azoles:

Triadimefon,Ketoconazole, CyproconazoleFluconazole, Itraconazole

Craniofacial malformations

Control Azoles

Control

Azoles

triadimefon 25-250 µMtriadimenol 12.5-125 µMflusilazole 6.25-25 µMcyproconazole 15.625-250µMfluconazole 125-500 µMtebuconazole 31.25-500µMimazalil 5-10-50-100 µMketoconazole 5-10-50-100 µM

Fusion and reduction of the1° and 2° branchial arches

In vitro tested azoles

Normal Neural Crest cell migration Abnormal 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 perspectivesNext 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.