Fig. 4

Nicotine-induced apoptosis in hair cells within neuromasts. Nicotine-induced apoptosis was confirmed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. (A) Green dots represent hair cells. Apoptotic cells appear as light red dots in the merged image obtained by fluorescent microscopy after the TUNEL reaction. All images were obtained using transgenic (brn3c:EGFP) zebrafish at 120 hours post-fertilization. Scale bar=10 μm (×40). (B) The average number of TUNEL-positive cells was measured in each neuromast (supraorbital [SO1, SO2], otic [O1], and occipital [OC1]; total n=50). At a concentration of 40 μM, the fish were almost all dead and statistical analysis was unavailable. The number of TUNEL-positive cells was significantly increased in nicotine-exposed groups (P<0.001, one-way analysis of variance). Only statistically significant pair-wise comparisons in post-hoc analysis are shown. *Statistically significant (P<0.05).