In vivo assessment of hair cell damage and developmental toxicity caused by gestational caffeine exposure using zebrafish (Danio 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 μM, 125 μM, 250 μM, and 500 μM) 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 μM 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 mitochondrial damage in high caffeine concentrations (250, 500 μM) (p < 0.01, DASPEI assay).Morphologic abnormalities were significantly increased in high caffeine concentrations (250 or 500 μM) 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 μM caffeine. Rapid hatching was present at 48 h (control; 46.6%, 500 μM caffeine; 100%). In conclusion, gestational caffeine exposure caused significant hair cell damage and developmental toxicities in zebrafish at early developmental stages.