In vivo assessment of hair cell damage and developmental toxicity caused by gestational caffeine exposure using zebrafish (Dario rerio) models

The aim of the present study was to evaluate hair cell damage and associated developmental toxicity caused by gestational caffeine exposure. We exposed embryos to various caffeine concentrations (25 mu M, 125 mu M, 250 mu M, and 500 mu M) and evaluated developmental toxicity of the embryos at 72 and 120 h and hair cell damage at 120 h after fertilization. The average number of total hair cells within four neuromasts exposed to various concentrations of caffeine was compared with that of the control group. To seek the underlying mechanisms, TUNEL and DASPEI assay were carried out to evaluate hair cell apoptosis and mitochondrial damage, respectively. Morphologic abnormality, mortality, hatching rate, and heart rate were also evaluated. Caffeine induced significant hair cell damage compared with control group (p < 0.01, control; 35.64 +/- 10.48 cells, 500 mu M caffeine; 23.32 +/- 12.14 cells, n = 25-30). Significant increase in the hair cell apoptosis was confirmed in a dose-dependent manner (p < 0.01, TUNEL assay) and the mitochondria! damage in high caffeine concentrations (250, 500 mu M) (p < 0.01, DASPEI assay). Morphologic abnormalities were significantly increased in high caffeine concentrations (250 or 500 mu M) for body shape, notochord, and heart at both 3-, and 5-dpf. The control group exhibited 3.3% mortality which increased up to 11.6% at 500 mu M caffeine. Rapid hatching was present at 48 h (control; 46.6%, 500 mu M caffeine; 100%). In conclusion, gestational caffeine exposure caused significant hair cell damage and developmental toxicities in zebrafish at early developmental stages.