Effect of N′-nitrosonornicotine (NNN) on murine palatal fusion in vitro

Add time:08/19/2019    Source:sciencedirect.com

Maternal smoking has been linked to an increased risk for orofacial clefts. N′-nitrosonornicotine (NNN) is one of the tobacco-specific nitrosamines that has been shown to be linked to the deleterious effects of tobacco and could be linked to the formation of cleft palate birth defects. The effect of NNN on palatal fusion was examined using an in vitro organ culture model of palatal development. The organ cultures were exposed to NNN (0.01, 0.1, 1, 10 and100 mM) and the effects on palatal development characterized at defined points. Palatal fusion was evaluated at embryonic day 13 (E13) + 72 h by characterizing the remaining medial edge epithelium (MEE) and determining the extent of fusion compared to controls. The NNN-treated group (1 mM) had more MEE remaining in the palatal midline than the untreated group at E13 + 72 h (P < 0.05). Changes in cell proliferation in the MEE resulting from NNN exposure were examined by BrdU incorporation in replicating DNA. Changes in the pattern of MEE cell death were examined by TUNEL. BrdU incorporation and TUNEL staining showed that the NNN (1 mM)-treated palates had more MEE cell proliferation and less apoptosis than the untreated-palates at E13 + 24 h (P < 0.05). The mechanism altered by NNN was further evaluated by characterizations of extracellular signal-regulated kinase (ERK) 1/2, p38 and c-jun amino-terminal kinase (JNK). NNN at 1 mM induced ERK1/2 phosphorylation, but reduced p38 phosphorylation (P < 0.05, P < 0.01, respectively) in the MEE. The results suggest that NNN inhibited palatal fusion by effects on cell proliferation and MEE cell death.

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