Pathogenic pathways in fluconazole-induced branchial arch malformations.
BACKGROUND
A widely-used antimycotic agent, bis-triazole fluconazole (FLUCO), is able to produce abnormalities to the branchial apparatus (hypoplasia, agenesis, and fusion) in postimplantation rodent embryos cultured in vitro. The branchial apparatus is a complex and transient structure in vertebrate embryos and is essential for the development of the face skeleton. Branchial arch mesenchyme is formed by two different cellular populations: paraxial mesenchyme and ectomesenchyme, which originate from rhombencephalic neural crest cell (NCC) migration. We investigated the possible pathogenic pathways involved in FLUCO-related branchial arch abnormalities. Perturbations in physiological apoptosis, cell proliferation, NCC migration and branchial mesenchyme induction have been considered.


METHODS
Rat embryos (9.5-day postcoitum; 1-3 somites) were exposed in vitro to 0 or 500 microM FLUCO. After 24, 36, or 48 hr of culture, embryos were examined for apoptosis (acridine orange method) and cell proliferation (BrdU incorporation and detection method). Rhombencephalic NCC migration was analyzed using immunostaining of NCC (using anti-CRABP antibodies) and the extracellular matrix (using anti-fibronectin antibodies). The differentiative capability of the branchial mesenchymes was investigated using anti-endothelin and anti-endothelin-receptor antibodies.


RESULTS
During the whole culture period, no alterations in physiological apoptosis, cell proliferation, and mesenchymal cell induction were observed in FLUCO-exposed embryos in comparison to controls. On the contrary, severe alterations in NCC migration pathways were observed in FLUCO-exposed embryos.


CONCLUSIONS
The findings suggest that FLUCO produces teratogenic effects by interfering with the cellular and molecular mechanisms that control NCC migration.
Menegola
Elena
E
Department of Biology, University of Milan, Milan, Italy.
Broccia
Maria L
ML
Di Renzo
Francesca
F
Giavini
Erminio
E
D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D001934:Branchial Region / Q000002:abnormalities* / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D005314:Embryonic and Fetal Development / Q000187:drug effects; D019332:Endothelin-1 / Q000032:analysis; D005260:Female; D005326:Fetal Proteins / Q000032:analysis; D015725:Fluconazole / Q000494:pharmacology / Q000633:toxicity*; D009432:Neural Crest / Q000187:drug effects; D009924:Organ Culture Techniques; D011247:Pregnancy; D051381:Rats; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin / Q000032:analysis
D000935:Antifungal Agents; D019332:Endothelin-1; D005326:Fetal Proteins; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin; D015725:Fluconazole