Molecular mechanism of teratogenic effects induced by the fungicide triadimefon: Study of the expression of TGF-beta mRNA and TGF-beta and CRABPI proteins during rat in vitro development

Azole derivatives are teratogenic in rats and mice in vitro and in vivo. The postulated mechanism for the dysmorphogenetic effects is the inhibition of retinoic acid (RA)-degrading enzyme CYP26. Azole-related abnormalities are confined to structures controlled by RA, especially the neural crest cells, hindbrain, cranial nerves, and craniolfacial structures, through a complex signal cascade. The aim of this work is to study the expression of signal molecules activated by RA (TGF-beta s) or involved in the modulation of cellular RA concentrations (CRABPI). E9.5 (9.5 day post cotium old embryos) rat embryos, exposed in vitro to triadimefon (FON) for 24 h, were examined or cultured in normal serum for extra 4, 16, and 24 h. RT-PCR was performed to quantify TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta RI, TGF-beta RII, and TGF-beta RIII mRNA in the hindbrain after 24 h of culture. TGF-beta 1, TGF-beta 2, and TGF-beta R1 were found significantly decreased by FON exposure, and consequently their protein expression was analyzed by Western blot and immunochemistry. In both controls and FON-exposed embryos, TGF-beta 1 and TGF-beta R1 were detected at 24 and 24 +4 h; TGF-beta 2 was present only at 24 h. Only TGF-beta 1 was expressed at the level of hindbrain and branchial tissues. After quantization, TGF-beta 1 was reduced in the FON group. The expression of CRABPI was observed at all developmental stages. However, in FON-exposed embryos, it was increased at 24 and 24+4 h. The hindbrain distribution of CRABPI-positive cells was abnormal in FON-exposed embryos. The results show that the two RA-related molecules (TGF-beta 1 and CRABPI) are altered by FON exposure in vitro. (C) 2008 Elsevier Inc. All rights reserved.